Hydrogels for vocal cord and soft tissue augmentation and repair

ABSTRACT

The present invention provides hydrogels and compositions thereof for vocal cord repair or augmentation, as well as other soft tissue repair or augmentation (e.g., bladder neck augmentation, dermal fillers, breast implants, intervertebral disks, muscle-mass). The hydrogels or compositions thereof are injected into the superficial lamina propria or phonatory epithelium to restore the phonatory mucosa of the vocal cords, thereby restoring a patient&#39;s voice. In particular, it has been discovered that hydrogels with an elastic shear modulus of approximately 25 Pa are useful in restoring the pliability of the phonatory mucosa. The invention also provides methods of preparing and using the inventive hydrogels.

RELATED APPLICATIONS

The present application claims priority under 35 U.S.C. §119(e) to U.S.provisional patent application, U.S. Ser. No 61/094,237, filed Sep. 4,2008, which is incorporated herein by reference.

BACKGROUND OF THE INVENTION

The vocal folds are the primary vibratory tissues essential for voiceproduction. In humans, there are two vocal folds, each consisting of astratified squamous epithelium, which encapsulates the lamina propria(LP) and the vocalis muscle (Hirano, Phonosurgery: Basic and ClinicalInvestigations. Otologia (Fukuoka), 1975. 21: p. 239-442; Hirano,Structure of the vocal fold in normal and diseased states: anatomicaland physical studies. Proceedings of the Conference on the Assessment ofVocal Pathology; The American Speech-Language-Hearing Association,1981.11: p. 11-27; each of which is incorporated herein by reference).The lamina propria is a soft tissue that can be roughly divided intothree layers: superficial, intermediate, and deep. The vocal ligament iscomprised of the intermediate and deep layers of the lamina propria, andthe vocal muscle is situated deep to the vocal ligament. See FIGS 1 a-1b. Vocal cord mucosa (i.e., the superficial lamina propria and theoverlying epithelium) has long been recognized as the key vibratorylayer critical for normal phonation (Bishop, J., Experimental Researchesinto the Physiology of the Human Voice. The London & EdinburghPhilosophical Magazine & Journal of Science, 1836; incorporated hereinby reference). The superficial lamina propria (SLP) is a relativelyacellular and pliable soft tissue and is the key constituent of thephonatory mucosa responsible for vibration. It must be identified,assessed, and preserved in most voice surgical procedures (Zeitels, S.M., Hillman, R. E., Franco, R. A., Bunting, G., Voice and TreatmentOutcome from Phonosurgical Management of Early Glottic Cancer. Annals ofOtology, Rhinology and Laryngology, 2002. 111(Supplement 190): p. 1-20;Zeitels, S. M., Hillman, R. E., Desloge, R. B., Mauri, M., Doyle, P. B.,Phonomicrosurgery in Singers & Performing Artists: Treatment Outcomes,Management Theories, & Future Directions. Annals of Otology, Rhinology,& Laryngology, 2002. 111(Supplement 190): p.21-40; Zeitels, S. M.,Healy, G. B., Laryngology and Phonosurgery. New England Journal ofMedicine, 2003. 349(9): p. 882-92; each of which is incorporated hereinby reference). Diminished pliability of vocal cord mucosa typicallyresults in permanent hoarseness (Zeitels, S. M., Hillman, R. E., Franco,R. A., Bunting, G., Voice and Treatment Outcome from PhonosurgicalManagement of Early Glottic Cancer. Annals of Otology, Rhinology andLaryngology, 2002. 111(Supplement 190): p. 1-20; Zeitels, S. M.,Hillman, R. E., Desloge, R. B., Mauri, M., Doyle, P. B.,Phonomicrosurgery in Singers & Performing Artists: Treatment Outcomes,Management Theories, & Future Directions. Annals of Otology, Rhinology,& Laryngology, 2002. 111(Supplement 190): p. 21-40; Zeitels, S. M.,Healy, G. B., Laryngology and Phonosurgery. New England Journal ofMedicine, 2003. 349(9): p. 882-92; each of which is incorporated hereinby reference). This diminished pliability can result from benign tumors,malignant tumors, diseases, and intubation (e.g., for anesthesia orprolonged intensive care unit respiratory support). Even self-inducedinsult to the vocal folds in the form of excessive speaking(phonotrauma) over an extended period of time or environmental insultssuch as smoke, alcohol, or stomach acid from reflux disease can resultin stiffening and scarring of the superficial lamina propria. One of themost common defects is the depostion of subepithelial type I collagen inthe vocal cord phonatory mucosa resulting in scarring of the SLP.

There have been attempts to restore the SLP using autograft fat tissue,but such attempts have had limited success (Zeitels, S. M., Sulcus,Scar, Synechia, and Web, in Atlas of Phonomicrosurgery and OtherEndolaryngeal Procedures for Benign and Malignant Disease. 2001,Singular: San Diego. p. 133-151; incorporated herein by reference).Currently, there has been no clinically feasible and reproducible methodthat has been demonstrated to restore SLP pliability, thereby repairingphonatory mucosal stiffness and eliminating or reducing the associatedhoarseness. To date there is simply no synthetic material or autograftthat will restore lost pliability to phonatory mucosa to resolve thedisordered vocal cord vibration and the associated permanent hoarseness(Zeitels, S. M., Blitzer, A., Hillman, R. E., Anderson, R. R., Foresightin Layngology and Laryngeal Surgery: A 2020 Vision. Ann Otol RhinolLaryngol, 2007. 116 (Supplement 198): p. 1-16; incorporated herein byreference). All current strategies just change the shape and position ofthe dysfunctional vocal fold to achieve better valvular closure(Zeitels, S. M., Jarboe, J., Franco, R. A., Phonosurgical Reconstructionof Early Glottic Cancer. Laryngoscope, 2001. 111: p. 1862-1865; Kriesel,K. J., Thibeault, S. L., Chan, R. W., Suzuki, T., VanGroll, P. J.,Bless, D. M., Ford, C. N., Treatment of vocal fold scarring: Rheologicaland histological measures of homologous collagen matrix. Annals ofOtology, Rhinology, and Laryngology, 2002. 111: p.884-889; each of whichis incorporated herein by reference). These techniques enhance voiceformation by diminishing aerodynamic incompetency, a surgical maneuverthat was done for the first time almost a century ago (Brunings, W.,Eine neue Behandlungsmethode der Rekurrenslahmungen. Verhandl DeutschVereins Deutscher Laryngologen, 1911. 18:93-151; which is incorporatedherein by reference). These strategies only achieve severely limitedresults and do not address the anatomic, physiologic, and/orbiomechanical deficit.

SUMMARY OF THE INVENTION

The present invention provides a system for repairing the pliability ofthe phonatory mucosa of a subject using hydrogels. The system involvesinserting or injecting a hydrogel or other material into thesubepithelial phonatory mucosal region where the superficial laminapropria is missing (e.g., after successful treatment of vocal cordcancer) or has diminished functional vibratory capacity (e.g., chronichoarseness from voice overuse and/or smoking). This is done to restorephonatory mucosal pliability and normal vocal cord vibration, therebyreducing stiffness and the associated hoarseness. It has been discoveredthat hydrogels with an elastic shear modulus of approximately 25 Pa areparticularly useful in the inventive system for vocal cord repair. Thehydrogels useful in the present invention are typicallysemi-interpenetrating networks formed when one polymer is cross-linkedwith itself in the presence of a non-crosslinkable polymer. Theinvention provides novel hydrogel compositions useful in the inventivesystem for vocal cord repair as well as methods and apparatuses fordelivering the hydrogel to a space created just under or within thesuperficial lamina propria. The injected hydrogel acts to augment thephonatory mucosa allowing this tissue to vibrate and produce sound.Other materials such as, but not limited to, viscosupplements (e.g.,HYALGAN® (sodium hyaluronate), SYNVISC® (Hylan G-F 20), ORTHOVISC® (highmolecular weight hyaluronan)) and dermal fillers (e.g., RESTYLANE®(hyaluronic acid), PERLANE® (hyaluronic acid), HYLAFORM® (stabilizedhyaluronic acid), RADIESSE® (calcium hydroxylapatite microspheres in awater-based gel)) may also be used for vocal cord repair. As will beappreciated by one of skill in the art, the hydrogels described hereinare also useful in repairing and/or augmenting other soft tissues. Forexample, the hydrogels may be used as deep vocal cord implants(paraglottic muscles) for medialization (e.g., paralytic dysphonia),dermal fillers, breast implants, bladder neck implants for incontinence,intervertebral disks, muscle mass, facial contouring, and joint fluid.It may also be used for packing wounds or incisions including orificessuch as the nose and ear.

In one aspect, the invention provides novel hydrogels for use in thepresent system for vocal cord repair, or other soft tissue repair oraugmentation. These novel hydrogels may provide immediate reconstitutionof the vocal cord, which may be long-lasting or temporary, and thehydrogel may provide a substrate or scaffold to introduce cells (e.g.,pluripotent cells such as stem cells) or other biologically activeagents (e.g., drugs). For example, a dissolving hydrogel may bereinserted/reinjected in the operating room or the clinic every 2-3months, or every 6 months as needed by the subject, which is similar topatients who are administered periodic injections of Botox (botulinumtoxin) to treat spasmodic dysphonia. Hydrogels useful in the presentinvention are typically semi-interpenetrating networks of polymersalthough interpenetrating networks of polymers and one-componenthydrogels may also find use in the present invention. The hydrogels havean elastic shear modulus ranging from approximately 15 Pa toapproximately 35 Pa. In certain embodiments, the elastic shear modulusranges from approximately 20 Pa to approximately 30 Pa. In certainembodiments, the elastic shear modulus of the hydrogel is approximately25 Pa. In certain embodiments, the hydrogel comprises acrylatedpolyethylene glycol and another water-soluble non-crosslinkable polymer(e.g., polyethylene glycol, proteins, hyaluronic acid, collagen,polylysine, dextran, alginates, gelatin, elastin, cellulose, etc.). Incertain embodiments, the hydrogel comprises a cross-linkable protein orpeptide (e.g., cross-linkable derivatives of elastin-like peptides,collagen-mimetic peptides, collagen-related peptides, polylysine) or across-linkable polysaccharide (e.g., cross-linkable derivatives ofhyaluronic acid, methyl cellulose, dextran, alginate, etc.). In certainembodiments, the hydrogel comprises a cross-linkable elastomeric polymer(e.g., cross-linkable derivative of poly glycerol sebacate). Thecross-linkable peptide, protein, or polysaccharide may include anacrylate moiety for cross-linking. Various polymers, molecular weights,extent of cross-linking, concentrations, cross-linkable moieties, andratio of crosslinkable versus non-crosslinkable polymers may be utilizedin the present invention. In certain embodiments, the crosslinkablepolymer component of the hydrogel is cross-linked usingphoto-crosslinking (e.g., using UV light). In certain embodiments, thecrosslinkable polymer component of the hydrogel is cross-linked using afree-radical reaction. Such a free radical reaction may be initiatedusing light, heat, or a biological or chemical catalyst. In certainembodiments, the free radical reaction is initiated using heat. Incertain embodiments, the free radical reaction is initiated using light(e.g., UV light). The hydrogel is typically biocompatible and is notreadily biodegradable so that it has an extended half-life in vivo atthe site of injection. Biologically active agents, such as cells,polynucleotides, proteins, pharmaceutical agents, etc., ormicroparticles, nanoparticles, or other drug delivery devices containingany of these biologically active agents may be added to the hydrogel.

In yet another aspect, the invention provides kits useful in treating apatient in need of vocal cord repair. The kit may include all or asubset of all the components necessary for treating a patient. The kitsmay include, for example, the hydrogel, components of the hydrogel,cross-linking reagent, UV lamp, syringe, needle, anesthetics,antibiotics, etc. In certain embodiments, the components of the kit aresterilely packaged for convenient use by the surgeon or other healthcare professional. The kit may also include instructions foradministering the hydrogel. The kit may provide the necessary componentsfor a single use. The kit may also include packaging and information asrequired by a governmental regulatory agency that regulatespharmaceuticals and/or medical devices.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1. FIG. 1 a is a cephalad view of the laryngeal introitus and vocalfolds from the oropharynx. FIG. 1 b shows a coronal section of the vocalfolds showing their layered micro-structure during phonation at lowpitch and high pitch. The top of each figure is cephalad anatomically.

FIG. 2. Dependence of elastic shear modulus of the hydrogels on thefraction of polyethylene glycol-diacrylate (PEG-DA) present in theinterpenetrating network. The second component of the interpenetratingnetwork is polyethylene glycol (PEG). The elastic shear modulus stronglydepends not only on the fraction of PEG-DA but also on the molecularweights of the components.

FIG. 3. Differentiation between material stiffness based on theirability to produce vocal fold excursion in a cow larynx model.

FIG. 4. Vocal fold excursion in a cow larynx model upon implantation ofdifferent materials.

FIG. 5. Percentage of amplitude ratio of the PEG30-injected vocal foldto the non-injected vocal fold as a function of time measured in vivousing a canine model.

FIG. 6. Slow release of rapamycin from PLGA (50:50; 75:25) nanoparticlesin buffer.

DEFINITIONS

“Anti-inflammatory agent,” as used herein, refers to any substance thatinhibits one or more signs or symptoms of inflammation.

“Anti-fibrotic agent,” as used herein, refers to any substance thatinhibits fibrosis. An exemplary anti-fibrotic agent is rapamycin.

An “aqueous medium” as used herein means a liquid medium containingwater and, optionally, one or more water-miscible solvents (e.g.,dimethylformamide (DMF), dimethylsulfoxide (DMSO), and hydrocarbylalcohols, diols, or glycerols). An aqueous medium may contain at least50%, 60%, 70%, 80%, 90% or more water by volume. It will be appreciatedthat an aqueous medium may contain a variety of substances dissolved,dispersed, or suspended therein.

The term “approximately” in reference to a number generally includesnumbers that fall within a range of 5% in either direction of the number(greater than or less than the number) unless otherwise stated orotherwise evident from the context (except where such number wouldexceed 100% of a possible value).

“Biocompatible” refers to a material that is substantially nontoxic to arecipient's cells in the quantities and at the location used, and alsodoes not elicit or cause a significant deleterious or untoward effect onthe recipient's body at the location used, e.g., an unacceptableimmunological or inflammatory reaction, unacceptable scar tissueformation, etc.

“Biocomparable” refers to a material that does not result indeterioration of normal function of residual or partially functioningtissue in the region in which the material is placed.

“Biodegradable” means that a material is capable of being broken downphysically and/or chemically within cells or within the body of asubject, e.g., by hydrolysis under physiological conditions and/or bynatural biological processes such as the action of enzymes presentwithin cells or within the body, and/or by processes such asdissolution, dispersion, etc., to form smaller chemical species whichcan typically be metabolized and, optionally, used by the body, and/orexcreted or otherwise disposed of. For purposes of the presentinvention, a polymer or hydrogel whose molecular weight decreases overtime in vivo due to a reduction in the number of monomers is consideredbiodegradable. In certain embodiments, the hydrogel useful in vocal cordrepair is not substantially biodegradable.

A “biologically active agent” is any compound or agent, or itspharmaceutically acceptable salt, which possesses a desired biologicalactivity, for example therapeutic, diagnostic, and/or prophylacticproperties in vivo. It is to be understood that the agent may need to bereleased from the hydrogel in order for it to exert a biologicalactivity. Biologically active agents include, but are not limited to,therapeutic agents as described herein. Biologically active agents maybe, without limitation, small molecules, peptides or polypeptides,immunoglobulins, e.g., antibodies, nucleic acids, cells, tissueconstructs, etc. Without limitation, hormones, growth factors, drugs,cytokines, chemokines, clotting factors and endogenous clottinginhibitors, etc., are biologically active agents.

The term “crosslinked” as used herein describes a polymer with at leastone covalent bond that is not found in the repeating units of thepolymer or found between repeating units of the polymer. Thecrosslinking bonds are typically between individual strands or moleculesof the polymer; however, intramolecular crosslinking to form macrocyclicstructures may also occur. The crosslinks are formed between any twofunctional groups of the polymer (e.g., at the ends, on the side chains,etc.). In certain embodiments, the crosslinks are formed betweenterminal acrylate units of the polymers. Also, any type of covalent bondmay form the crosslink (e.g., carbon-carbon, carbon-oxygen,carbon-nitrogen, oxygen-nitrogen, sulfur-sulfur, oxygen-phosphorus,nitrogen-nitrogen, oxygen-oxygen, etc.). The resulting crosslinkedmaterial may be branched, linear, dendritic, etc. In certainembodiments, the crosslinks form a 3-D network of crosslinks. Thecrosslinks may be formed by any chemical reaction that results in thecovalent bonds. Typically, the crosslinks are created by free radicalinitiated reactions, for example, with a photoinitiator or thermalinitiator.

The term “endoscope” or “laryngoscope” means an instrument used todirect the placement of the hydrogel in the correct layer of the vocalfold. There are generally three types of laryngoscopes. A rigidtelescope with angulated optics similar to a laryngeal mirror may beplaced through the mouth to the oropharynx to view the vocal cords. Aflexible laryngoscope is passed through the nose into the pharynx toview the vocal cords. A direct laryngoscope is comprised of a rigidspatula or tubular speculum, which is passed through the patient'smouth, typically used during an unconscious state, for visualizing andinstrumenting the vocal folds during endoscopic laryngeal surgery or forintubating a patient for general anesthesia or airway support.

A “hydrogel” is a three-dimensional network comprising hydrophilicpolymers that contains a large amount of water. A hydrogel may, forexample contain 30%, 40%. 50%, 60%, 70%, 80%, 90%, 95%, or an evengreater amount of water on a w/w basis. A “hydrogel precursor” is apolymer that is at least partly soluble in an aqueous medium and iscapable of becoming crosslinked to form a hydrogel.

“Interpenetrating network” refers to any material with a network ofpolymers where two polymers are cross-linked in the presence of eachother. Both polymers are cross-linkable, and each forms its own networkby cross-linking with itself but not with the other polymer. Typically,the two polymers are synthesized and/or cross-linked in the presence ofeach other, the polymers have similar kinetics, and the two polymers arenot dramatically phase separated.

The terms “polynucleotide”, “nucleic acid”, or “oligonucleotide” referto a polymer of nucleotides. The terms “polynucleotide”, “nucleic acid”,and “oligonucleotide”, may be used interchangeably. Typically, apolynucleotide comprises at least two nucleotides. DNAs and RNAs arepolynucleotides. The polymer may include natural nucleosides (i.e.,adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine,deoxythymidine, deoxyguanosine, and deoxycytidine), nucleoside analogs(e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine,3-methyl adenosine, C5-propynylcytidine, C5-propynyluridine,C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-methylcytidine,7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine,O(6)-methylguanine, and 2-thiocytidine), chemically modified bases,biologically modified bases (e.g., methylated bases), intercalatedbases, modified sugars (e.g., 2′-fluororibose, 2′-methoxyribose,2′-aminoribose, ribose, 2′-deoxyribose, arabinose, and hexose), ormodified phosphate groups (e.g., phosphorothioates and 5′-Nphosphoramidite linkages). Enantiomers of natural or modifiednucleosides may also be used. Nucleic acids also include nucleicacid-based therapeutic agents, for example, nucleic acid ligands, siRNA,short hairpin RNA, antisense oligonucleotides, ribozymes, aptamers, andSPIEGELMERS™, oligonucleotide ligands described in Wlotzka, et al.,Proc. Natl. Acad. Sci. USA, 2002, 99(13):8898, the entire contents ofwhich are incorporated herein by reference.

A “polypeptide”, “peptide”, or “protein” comprises a string of at leastthree amino acids linked together by peptide bonds. The terms“polypeptide”, “peptide”, and “protein”, may be used interchangeably.Peptide may refer to an individual peptide or a collection of peptides.Inventive peptides preferably contain only natural amino acids, althoughnon natural amino acids (i.e., compounds that do not occur in nature butthat can be incorporated into a polypeptide chain) and/or amino acidanalogs as are known in the art may alternatively be employed. Also, oneor more of the amino acids in a peptide may be modified, for example, bythe addition of a chemical entity such as a carbohydrate group, aphosphate group, a farnesyl group, an isofarnesyl group, a fatty acidgroup, a linker for conjugation, functionalization, or othermodification, etc. In one embodiment, the modifications of the peptidelead to a more stable peptide (e.g., greater half-life in vivo). Thesemodifications may include cyclization of the peptide, the incorporationof D-amino acids, etc. None of the modifications should substantiallyinterfere with the desired biological activity of the peptide.

The terms “polysaccharide” and “carbohydrate” may be usedinterchangeably. Most carbohydrates are aldehydes or ketones with manyhydroxyl groups, usually one on each carbon atom of the molecule.Carbohydrates generally have the molecular formula C_(n)H_(2n)O_(n). Acarbohydrate may be a monosaccharide, a disaccharide, trisaccharide,oligosaccharide, or polysaccharide. The most basic carbohydrate is amonosaccharide, such as glucose, sucrose, galactose, mannose, ribose,arabinose, xylose, and fructose. Disaccharides are two joinedmonosaccharides. Exemplary disaccharides include sucrose, maltose,cellobiose, and lactose. Typically, an oligosaccharide includes betweenthree and six monosaccharide units (e.g., raffinose, stachyose), andpolysaccharides include six or more monosaccharide units. Exemplarypolysaccharides include starch, glycogen, and cellulose. Carbohydratesmay contain modified saccharide units such as 2′-deoxyribose wherein ahydroxyl group is removed, 2′-fluororibose wherein a hydroxyl group isreplace with a fluorine, or N-acetylglucosamine, a nitrogen-containingform of glucose. (e.g., 2′-fluororibose, deoxyribose, and hexose).Carbohydrates useful in the present invention may be linear or branched.Carbohydrates may exist in many different forms, for example,conformers, cyclic forms, acyclic forms, stereoisomers, tautomers,anomers, and isomers.

“Semi-interpenetrating network” refers to a network of polymers whereone polymer is cross-linked with itself in the presence of anon-crosslinkable polymer.

“Small molecule” refers to organic compounds, whethernaturally-occurring or artificially created (e.g., via chemicalsynthesis) that have relatively low molecular weight and that are notproteins, polypeptides, or nucleic acids. Small molecules are typicallynot polymers with repeating units. In certain embodiments, a smallmolecule has a molecular weight of less than about 1500 g/mol. Also,small molecules typically have multiple carbon-carbon bonds and may havemultiple stereocenters and functional groups.

“Solubility” refers to the amount of a substance that dissolves in agiven volume of solvent at a specified temperature and pH, e.g., to forma saturated solution. Solubility may be determined, for example, usingthe shake-flask solubility method (ASTM: E 1148-02, Standard Test Methodfor Measurements of Aqueous Solubility, Book of Standards Volume 11.05).Solubility may be determined at a pH between 3.0 and 9.0, e.g., between4.0 and 8.0, between 5.0 and 8.0, between 6.0 and 8.0, e.g., between 6.5and 7.6, e.g., between 6.8-7.4, e.g., 7.0, or any intervening value ofthe foregoing ranges. Solubility may be tested at a temperature ofbetween 20 and 40° C., e.g., approximately 25-37° C., e.g.,approximately 37° C., or any intervening value of th foregoing ranges.For example, solubility may be determined at approximately pH 7.0-7.4and approximately 37° C.

“Subject,” as used herein, refers to an individual to whom an agent isto be delivered, e.g., for experimental, diagnostic, and/or therapeuticpurposes. Preferred subjects are mammals, particularly domesticatedmammals (e.g., dogs, cats, etc.), primates, or humans. A subject underthe care of a physician or other health care provider may be referred toas a “patient.”

“Pharmaceutical agent,” also referred to as a “drug,” is used herein torefer to an agent that is administered to a subject to treat a disease,disorder, or other clinically recognized condition that is harmful tothe subject, or for prophylactic purposes, and has a clinicallysignificant effect on the body to treat or prevent the disease,disorder, or condition. Therapeutic agents include, without limitation,agents listed in the United States Pharmacopeia (USP), Goodman andGilman's The Pharmacological Basis of Therapeutics, 10^(th) Ed., McGrawHill, 2001; Katzung, B. (ed.) Basic and Clinical Pharmacology,McGraw-Hill/Appleton & Lange; 8th edition (Sep. 21, 2000); Physician 'sDesk Reference (Thomson Publishing), and/or The Merck Manual ofDiagnosis and Therapy, 17^(th) ed. (1999), or the 18^(th) ed (2006)following its publication, Mark H. Beers and Robert Berkow (eds.), MerckPublishing Group, or, in the case of animals, The Merck VeterinaryManual, 9^(th) ed., Kahn, C. A. (ed.), Merck Publishing Group, 2005.

“Viscosity” refers to a measurment of the thickness or resistance toflow of a liquid at a given temperature. Viscosity may be determinedusing a variety of methods and instruments known in the art. Forexample, a polymer is first weighed and then dissolved in an appropriatesolvent. The solution and viscometer are placed in a constanttemperature water bath. Thermal equilibrium is obtained within thesolution. The liquid is then brought above the upper graduation mark onthe viscometer. The time for the solution to flow from the upper tolower graduation marks is recorded. Viscosity of a solution comprising apolymer may be determined in accordance with ASTM Book of Standards,Practice for Dilute Solution Viscosity of Polymers (ASTM D2857), Volume08.01, June 2005 or relevant ASTM standards for specific polymers.Solubility may be tested at a temperature of between 20 and 40° C.,e.g., approximately 25-37° C., e.g., approximately 37° C., or anyintervening value of the foregoing ranges. For example, solubility maybe determined at approximately pH 7.0-7.4 and approximately 37° C.

“Elastic shear modulus” of a material is a mathematical description of amaterial's tendency to be deformed elastically (i.e., non-permanently)when a force is applied parallel to one of its surfaces while itsopposite face experiences an opposing force (e.g., friction). Elasticshear modulus is calculated as the ratio of shear stress to shearstrain. For example, if a force of 1 N is applied tangentially (on thexy plane) to a surface of an area of 1 m² and produces a change in theshape by 1% (strain=0.01) in the xy plane, then the elastic shearmodulus of the material is 1/0.01=100 Pa.

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS OF THE INVENTION

The present invention stems from the recognition that the phonatorysubepithelial soft tissue of the vocal folds must be pliable so that itcan vibrate and produce sound. In a normal state, this is thesuperficial lamina propria (SLP). Based on this recognition, it has beendiscovered that injection of hydrogels into or underneath the phonatoryepithelium can improve phonation in patients with stiff and/or scarredvocal folds. Hydrogels useful in this procedure typically have anelastic shear modulus ranging from approximately 15 Pa to approximately35 Pa. The invention also provides novel hydrogels and compositionsthereof useful in vocal cord repair as well as other soft tissues (e.g.,skin, muscle, breast, bladder, intervertebral disks) of a patient.

Hydrogels

The present invention provides novel hydrogels for use in vocal cordrepair, or other soft tissue repair or augmentation. Hydrogels aresuperabsorbent natural or synthetic polymers. Hydrogels can contain upto 99% water by weight. It has been discovered that certain hydrogelsare useful in vocal cord repair. The hydrogel may include one or morepolymers. In certain embodiments, the hydrogel is a mixture ofcross-linked and/or uncross-linked polymers. In particular,semi-interpenetrating networks of polymers that form hydrogels have beenfound to be useful in vocal cord repair. Interpenetrating networks ofpolymers that form hydrogels and one-component hydrogels have also beenfound to be useful in vocal cord repair. Furthermore, it has beendiscovered that hydrogels with an elastic shear modulus ranging fromabout 15 Pa to about 35 Pa are particularly useful for restoring thepliability of the phonatory mucosa of the vocal cords. In certainembodiments, the elastic shear modulus of the hydrogel ranges from about20 Pa to about 30 Pa. In certain embodiments, the elastic shear modulusof the hydrogel is about 25 Pa.

In certain embodiments, the hydrogel only comprises one polymer. Incertain other embodiments, the hydrogel comprises more than one polymer.In certain embodiments, the hydrogel comprises two polymers. In certainembodiments, the hydrogel comprises three, four, five, or more polymers.A mixture of polymers allows one to tune the desired characteristics ofthe hydrogel. Any polymer may be used in preparing a hydrogel. Thepolymers of the hydrogel may be natural or synthetic. Typically, thepolymer(s) used in the hydrogel is at least somewhat water soluble.Examples of polymers useful in preparing hydrogels include, but are notlimited to, polycarbonates (e.g. poly(1,3-dioxan-2one)), polyanhydrides(e.g. poly(sebacic anhydride)), polyhydroxyacids (e.g.poly(β-hydroxyalkanoate)), polypropylfumerates, polycaprolactones,polyamides (e.g. polycaprolactam, polylysine, peptides made with D-aminoacids), polyacetals, polyethers, polyesters (e.g. polylactide,polyglycolide), poly(orthoesters), polycyanoacrylates, polyvinylalcohols, polyurethanes, polyphosphazenes, polyacrylates,polymethacrylates, polyureas, polysaccharides (e.g., hyaluronic acid,dextran, alginate, cellulose), polyamines, and co-polymers thereof.Examples of natural polymers include proteins, peptides (e.g.,elastin-like peptide, collagen-mimetic peptides, collagen-relatedpeptides), polysaccharides (e.g., hyaluronic acid, methyl cellulose,dextran, alginate), and nucleic acids.

In certain embodiments, the hydrogel is prepared using a polyol. Incertain embodiments, the hydrogel is prepared using a polyether (e.g.,polyethylene glycol, polypropylene glycol, poly(tetramethyleneether)glycol). In certain embodiments, the hydrogel comprisespolyethylene glycol. In certain embodiments, the hydrogel is preparedusing a polyether and another type of polymer. In certain particularembodiments, the hydrogel is prepared using polyethylene glycol andanother type of polymer. In certain embodiments, the hydrogel isprepared using a polyether and a protein. In certain embodiments, thehydrogel is prepared using a polyether and a polysaccharide. In certainembodiments, the hydrogel is prepared using a polyether and anotherpolyether. In certain embodiments, the hydrogel is prepared using apolyether and a polyol. In certain embodiments, the hydrogel is preparedusing at least two polyethers. In certain embodiments, the hydrogel isprepared using an acrylated version of polyethylene glycol and anothertype of polymer. In certain embodiments, the hydrogel is prepared usinga diacrylated version of polyethylene glycol and another type ofpolymer. In certain particular embodiments, the hydrogel is preparedusing poly(glycerol sebacate) and acrylated polyethylene glycol. Incertain particular embodiments, the hydrogel is prepared usinghyaluronic acid and acrylated polyethylene glycol. In certain particularembodiments, the hydrogel is prepared using methyl cellulose andacrylated polyethylene glycol. In certain particular embodiments, thehydrogel is prepared using dextran and acrylated polyethylene glycol. Incertain particular embodiments, the hydrogel is prepared using alginateand acrylated polyethylene glycol. In certain particular embodiments,the hydrogel is prepared using polylysine and acrylated polyethyleneglycol. In certain particular embodiments, the hydrogel is preparedusing poly(glycerol sebacate) and polyethylene glycol-diacrylate. Incertain particular embodiments, the hydrogel is prepared usinghyaluronic acid and polyethylene glycol-diacrylate. In certainparticular embodiments, the hydrogel is prepared using methyl celluloseand polyethylene glycol-diacrylate. In certain particular embodiments,the hydrogel is prepared using dextran and polyethyleneglycol-diacrylate. In certain particular embodiments, the hydrogel isprepared using alginate and polyethylene glycol-diacrylate. In certainparticular embodiments, the hydrogel is prepared using polylysine andpolyethylene glycol-diacrylate.

In certain embodiments, the hydrogel is prepared using a cross-linkablepeptide or protein and another type of polymer. In certain embodiments,the cross-linkable peptide is a cross-linkable version of elastin-likepeptides (ELP), collagen-mimetic peptides (CMP), or collagen-relatedpeptides (CRP). The peptide may include natural L-amino acids, unnaturalD-amino acids, or a combination thereof. When a peptide is made fromD-amino acids, the resulting peptide is typically less amenable tobiodegradation, in particular enzymatic degradation. In certainembodiments, the cross-linkable peptide includes an acrylated version ofthe peptide. Other cross-linkable moieties as described herein may alsobe used. The cross-linkable peptide may be combined with any otherpolymer as described herein. In certain embodiments, the cross-linkablepeptide is cross-linked in the presence of hyaluronic acid, collagen,gelatin, alginate, methyl cellulose, elastin, polylysine, or aderivative thereof. In certain embodiments, the hydrogel comprises asemi-interpenetrating network of acrylated ELP (D-peptide form) andhyaluronic acid. In certain embodiments, the hydrogel comprises asemi-interpenetrating network of acrylated ELP (L-peptide form) andhyaluronic acid. In certain embodiments, the hydrogel comprises asemi-interpenetrating network of acrylated ELP (D-peptide form) andcollagen. In certain embodiments, the hydrogel comprises asemi-interpenetrating network of acrylated ELP (L-peptide form) andcollagen. In certain embodiments, the hydrogel comprises asemi-interpenetrating network of acrylated ELP (D-peptide form) andpolylysine. In certain embodiments, the hydrogel comprises asemi-interpenetrating network of acrylated ELP (L-peptide form) andpolylysine. In certain embodiments, the hydrogel comprises asemi-interpenetrating network of acrylated ELP (D-peptide form) anddextran. In certain embodiments, the hydrogel comprises asemi-interpenetrating network of acrylated ELP (L-peptide form) anddextran. In certain embodiments, the hydrogel comprises asemi-interpenetrating network of acrylated ELP (D-peptide form) andalginate. In certain embodiments, the hydrogel comprises asemi-interpenetrating network of acrylated ELP (L-peptide form) andalginate. In certain embodiments, the hydrogel comprises asemi-interpenetrating network of acrylated polylysine and hyaluronicacid. In certain embodiments, the hydrogel comprises asemi-interpenetrating network of acrylated polylysine and dextran. Incertain embodiments, the hydrogel comprises a semi-interpenetratingnetwork of acrylated polylysine and alginate. In certain embodiments,the hydrogel comprises a semi-interpenetrating network of acrylatedpolylysine and elastin. In certain embodiments, the hydrogel comprises asemi-interpenetrating network of acrylated polylysine and collagen. Incertain embodiments, the hydrogel comprises a semi-interpenetratingnetwork of acrylated polylysine and polylysine. In certain embodiments,the hydrogel comprises a semi-interpenetrating network of acrylatedpolylysine and gelatin. In certain embodiments, the hydrogel willinclude only natural polymers. In certain embodiments, the hydrogel doesnot include polyethylene glycol or a derivative thereof.

In certain embodiments, the hydrogel is prepared using a cross-linkablepolysaccharide and another type of polymer. In certain embodiments, thecross-linkable polysaccharide is a water soluble polysaccharide. Incertain embodiments, the cross-linkable polysaccharide is a linearpolysaccharide. In other embodiments, the cross-linkable polysaccharideis a branched polysaccharide. In certain embodiments, the hydrogelcomprises a cross-linkable version of hyaluronic acid. In certainembodiments, the hydrogel comprises a cross-linkable version of methylcellulose or other cellulose derivative. In certain embodiments, thehydrogel comprises a cross-linkable version of dextran. In certainembodiments, the hydrogel comprises a cross-linkable version ofalginate. In certain embodiments, the cross-linkable polysaccharide isan acrylated version of a polysaccharide. Other cross-linkable moietiesas described herein may also be used. The cross-linkable polysaccharidemay be combined with any other polymer as described herein. In certainembodiments, the cross-linkable polysaccharide is cross-linked in thepresence of hyaluronic acid, collagen, dextran, gelatin, polylysine,alginate, methyl cellulose, elastin, or a derivative thereof. In certainembodiments, the hydrogel comprises a semi-interpenetrating network ofacrylated methyl cellulose and hyaluronic acid. In certain embodiments,the hydrogel comprises a semi-interpenetrating network of acrylatedmethyl cellulose and dextran. In certain embodiments, the hydrogelcomprises a semi-interpenetrating network of acrylated methyl celluloseand alginate. In certain embodiments, the hydrogel comprises asemi-interpenetrating network of acrylated methyl cellulose and elastin.In certain embodiments, the hydrogel comprises a semi-interpenetratingnetwork of acrylated methyl cellulose and collagen. In certainembodiments, the hydrogel comprises a semi-interpenetrating network ofacrylated methyl cellulose and polylysine. In certain embodiments, thehydrogel comprises a semi-interpenetrating network of acrylated methylcellulose and gelatin. In certain embodiments, the hydrogel comprises asemi-interpenetrating network of acrylated hyaluronic acid andhyaluronic acid. In certain embodiments, the hydrogel comprises asemi-interpenetrating network of acrylated hyaluronic acid and dextran.In certain embodiments, the hydrogel comprises a semi-interpenetratingnetwork of acrylated hyaluronic acid and alginate. In certainembodiments, the hydrogel comprises a semi-interpenetrating network ofacrylated hyaluronic acid and elastin. In certain embodiments, thehydrogel comprises a semi-interpenetrating network of acrylatedhyaluronic acid and collagen. In certain embodiments, the hydrogelcomprises a semi-interpenetrating network of acrylated hyaluronic acidand polylysine. In certain embodiments, the hydrogel comprises asemi-interpenetrating network of acrylated hyaluronic acid and gelatin.In certain embodiments, the acryalted hyaluronic acid is methacrylatedhyaluronic acid. In certain embodiments, the hydrogel comprises asemi-interpenetrating network of acrylated dextran and hyaluronic acid.In certain embodiments, the hydrogel comprises a semi-interpenetratingnetwork of acrylated dextran and dextran. In certain embodiments, thehydrogel comprises a semi-interpenetrating network of acrylated dextranand alginate. In certain embodiments, the hydrogel comprises asemi-interpenetrating network of acrylated dextran and elastin. Incertain embodiments, the hydrogel comprises a semi-interpenetratingnetwork of acrylated dextran and collagen. In certain embodiments, thehydrogel comprises a semi-interpenetrating network of acrylated dextranand polylysine. In certain embodiments, the hydrogel comprises asemi-interpenetrating network of acrylated dextran and gelatin. Incertain embodiments, the hydrogel comprises a semi-interpenetratingnetwork of acrylated alginate and hyaluronic acid. In certainembodiments, the hydrogel comprises a semi-interpenetrating network ofacrylated alginate and dextran. In certain embodiments, the hydrogelcomprises a semi-interpenetrating network of acrylated alginate andalginate. In certain embodiments, the hydrogel comprises asemi-interpenetrating network of acrylated alginate and elastin. Incertain embodiments, the hydrogel comprises a semi-interpenetratingnetwork of acrylated alginate and collagen. In certain embodiments, thehydrogel comprises a semi-interpenetrating network of acrylated alginateand polylysine. In certain embodiments, the hydrogel comprises asemi-interpenetrating network of acrylated alginate and gelatin. Incertain embodiments, the hydrogel will include only natural polymers. Incertain embodiments, the hydrogel includes only polysaccharides orderivatives of polysaccharides. In certain embodiments, the hydrogeldoes not include polyethylene glycol or a derivative thereof.

In certain embodiments, the hydrogel is prepared using a cross-linkableelastomeric polymer and another type of polymer. In certain embodiments,the hydrogel comprises a cross-linkable version of poly(glycerolsebacate) (PGS). In certain embodiments, the cross-linkablepolysaccharide is an acrylated version of an elastomeric polymer. Othercross-linkable moieties as described herein may also be used. Thecross-linkable elastomeric polymer may be combined with any otherpolymer as described herein. In certain embodiments, the cross-linkableelastomeric polymer is cross-linked in the presence of hyaluronic acid,collagen, gelatin, alginate, methyl cellulose, elastin, dextran,polylysine, or a derivative thereof. In certain embodiments, thecross-linkable elastomeric polymer is cross-linked in the presence ofpolyethylene glycol, poly(lactic acid), poly(glycolic acid),poly(lactic-co-glycolic acid), or a derivative thereof. In certainembodiments, the hydrogel comprises a semi-interpenetrating network ofacrylated PGS and hyaluronic acid. In certain embodiments, the hydrogelcomprises a semi-interpenetrating network of acrylated PGS and methylcellulose. In certain embodiments, the hydrogel comprises asemi-interpenetrating network of acrylated PGS and elastin. In certainembodiments, the hydrogel comprises a semi-interpenetrating network ofacrylated PGS and collagen. In certain embodiments, the hydrogelcomprises a semi-interpenetrating network of acrylated PGS and gelatin.In certain embodiments, the hydrogel comprises a semi-interpenetratingnetwork of acrylated PGS and dextran. In certain embodiments, thehydrogel comprises a semi-interpenetrating network of acrylated PGS andalginate. In certain embodiments, the hydrogel comprises asemi-interpenetrating network of acrylated PGS and polylysine. Incertain embodiments, the hydrogel comprises a semi-interpenetratingnetwork of acrylated PGS and polyethylene glycol (PEG). In certainembodiments, the hydrogel will include only natural polymers. In certainembodiments, the hydrogel does not include polyethylene glycol or aderivative thereof.

In certain embodiments, the hydrogel is a semi-interpenetrating networkof polymers formed when a polymer is crosslinked with itself in thepresence of a non-crosslinkable polymer. In certain embodiments, thecrosslinkable polymer is water soluble. In certain embodiments, thenon-crosslinkable polymer is water soluble. The water-soluble polymertypically has a minimum solubility of at least approximately 0.1 g ofpolymer per liter of water. In certain embodiments, the solubility ofthe polymer in water is at least approximately 0.5 g of polymer perliter of water. In certain embodiments, the solubility of the polymer inwater is at least approximately 1 g of polymer per liter of water. Incertain embodiments, the solubility of the polymer in water is at leastapproximately 5 g of polymer per liter of water. In certain embodiments,the solubility of the polymer in water is at least approximately 10 g ofpolymer per liter of water. The hydrogel may also include otherpolymers, which may be water soluble or not. Any of the polymersdescribed herein may be used to prepare semi-interpenetrating networksof polymers. In certain embodiments, a polyether is used in thepreparation of the semi-interpenetrating network of polymers. In certainembodiments, a polymer is modified to make it suitable forcross-linking. For example, functional groups suitable for cross-linking(e.g., acrylate moieties, vinyl moieties, alkenyl moieties, alkynylmoieties, methacrylate moieties, cyanoacrylate moieties) may be added tothe polymer.

The crosslinkable polymer component of the hydrogel may be any syntheticor natural polymer which is capable of being cross-linked. In certainembodiments, the cross-linkable polymer is a synthetic polymer. Incertain embodiments, the crosslinkable polymer is a natural polymer suchas a protein or carbohydrate. The polymer typically will include or maybe modified to include functional groups suitable for cross-linking suchas acrylates, methacrylates, alkenes, alkynes, carboxylic acids, amines,aldehydes, halides, azides, esters, thiols, diazirines, carbodiimides,imidoesters, azenes, strained rings such as epoxides or aziridines, etc.In certain embodiments, the polymer is an acrylated polyethylene glycol.For example, polyethylene glycol diacrylate, polyethylene glycoltriacrylate, etc. may be used as the crosslinkable polymer in thehydrogel. Other polymers besides polyethylene glycol may form thebackbone of the polymer. Other exemplary polymer backbones include, butare not limited to, polyesters, polyamines, polyethers, polyamides,polyureas, polyanhydrides, polyhydroxyacids, polypropylfumarates,polycaprolactones, polyacetals, poly(orthoesters), polyvinyl alcohol,polyurethanes, polyphosphazenes, and polycarbonates. In certainembodiments, the polymer backbone is polypropylene glycol. In certainembodiments, the polymer backbone is polybutylene glycol. In certainembodiments, the polymer is methacrylated rather than acrylated. Incertain embodiments, the polymer is cyanoacrylated. In otherembodiments, the polymer comprises vinyl moieties rather than acrylateor methacrylate moieties. In certain embodiments, the polymer comprisesazide moieties. In certain embodiments, the polymer comprises strainedrings. In certain embodiments, the polymer comprises an epoxide moiety.In certain embodiments, the polymer comprises an aziridine moiety. Incertain embodiments, the polymer comprises an amine. In certainembodiments, the polymer comprises an aldehyde. In certain embodiments,the polymer comprises a halogen. In certain embodiments, the polymercomprises an alkenyl moiety. In certain embodiments, the polymercomprises an alkynyl moiety. In certain embodiments, the polymercomprises a carboxylic acid. In certain embodiments, the polymercomprises an ester. In certain embodiments, the polymer comprises athiol. In certain embodiments, the polymer comprises a diazirine. Incertain embodiments, the polymer comprises a carbodiimide. In certainembodiments, the polymer comprises an imidoester. In certainembodiments, the polymer comprises an azene moiety. In certainembodiments, the polymer comprises a nitrene moiety.

The non-crosslinkable polymer component of the hydrogel may also besynthetic or natural. Typically, the non-crosslinkable polymer componentof the hydrogel is a water-soluble polymer. In certain embodiments, thenon-crosslinkable polymer is a synthetic polymer. In other embodiments,the non-crosslinkable polymer is a natural polymer. Exemplarynon-crosslinkable, water soluble polymers include, but are not limitedto, polyethers, polypeptides (e.g., polylysine, polyserine,polythreonine, polyglutamate, polyaspartate, polyhistidine,polyarginine), polysaccharides (e.g., alginates, dextran, cellulose,hyaluronic acid), polyamides, proteins (e.g., gelatin, elastin), andderivatives thereof. In certain embodiments, the non-crosslinkablepolymer is analogous to the crosslinkable polymer, for example, anon-acrylated polymer (e.g., polyethylene glycol) versus an acrylatedpolymer (e.g., polyethylene glycol diacrylate).

The physicochemical properties of the hydrogel may be varied by changingthe portion of crosslinkable polymer as compared to non-crosslinkablepolymer, molecular weights of either or both polymers, concentration ofpolymer, and extent of cross-linking.

The molecular weight of either polymer may range from approximately2,000 g/mol up to approximately 600,000 g/mol. In certain embodiments,the molecular weight of the polymer ranges from approximately 5,000g/mol to approximately 30,000 g/mol. In certain embodiments, themolecular weight of the polymer ranges from approximately 5,000 g/mol toapproximately 10,000 g/mol. In certain embodiments, the molecular weightof the polymer ranges from approximately 10,000 g/mol to approximately15,000 g/mol. In certain embodiments, the molecular weight of thepolymer ranges from approximately 10,000 g/mol to approximately 20,000g/mol. In certain embodiments, the molecular weight of the polymerranges from approximately 20,000 g/mol to approximately 30,000 g/mol. Incertain embodiments, the molecular weight of the polymer ranges fromapproximately 30,000 g/mol to approximately 40,000 g/mol. In certainembodiments, the molecular weight of the polymer ranges fromapproximately 40,000 g/mol to approximately 50,000 g/mol. In certainembodiments, the molecular weight of the crosslinkable polymer beforecross-linking is approximately 5,000 g/mol, approximately 10,000 g/mol,approximately 15,000 g/mol, approximately 20,000 g/mol, approximately25,000 g/mol, approximately 30,000 g/mol, approximately 35,000 g/mol,approximately 40,000 g/mol, approximately 45,000 g/mol, andapproximately 50,000 g/mol. In certain embodiments, the molecular weightof the non-crosslinkable polymer is approximately 5,000 g/mol,approximately 10,000 g/mol, approximately 15,000 g/mol, approximately20,000 g/mol, approximately 25,000 g/mol, approximately 30,000 g/mol,approximately 35,000 g/mol, approximately 40,000 g/mol, approximately45,000 g/mol, and approximately 50,000 g/mol. In certain embodiments,the molecular weight of the polymer ranges from approximately 50,000g/mol to approximately 100,000 g/mol. In certain embodiments, themolecular weight of the polymer ranges from approximately 100,000 g/molto approximately 200,000 g/mol. In certain embodiments, the molecularweight of the polymer ranges from approximately 200,000 g/mol toapproximately 300,000 g/mol. In certain embodiments, the molecularweight of the polymer is approximately 250,000 g/mol. In certainembodiments, the molecular weight of the polymer ranges fromapproximately 300,000 g/mol to approximately 400,000 g/mol. In certainembodiments, the molecular weight of the polymer ranges fromapproximately 400,000 g/mol to approximately 500,000 g/mol. In certainembodiments, the molecular weight of the polymer ranges fromapproximately 500,000 g/mol to approximately 600,000 g/mol.

Any ratio of crosslinkable to non-crosslinkable polymer may be used inthe inventive hydrogels. In certain embodiments, a nearly equal portionof each polymer component is used to prepare the hydrogel. In certainembodiments, the amount of one of the polymers is greater than theother. In certain embodiments, the amount of the non-crosslinkablepolyer is greater than the amount of the crosslinkable polymer. Incertain embodiments, the ratio of non-crosslinkable polymer tocrosslinkable polymer is about 10:90, about 20:80, about 30:70, about40:60, about 50:50, about 60:40, about 70:30, about 80:20, or about90:10. In certain particular embodiments, the ratio of non-crosslinkablepolymer to crosslinkable polymer is about 70:30. In certain particularembodiments, the ratio of non-crosslinkable polymer to crosslinkablepolymer is about 69:31. In certain particular embodiments, the ratio ofnon-crosslinkable polymer to crosslinkable polymer is about 71:29. Incertain particular embodiments, the ratio of non-crosslinkable polymerto crosslinkable polymer is about 72:28. In certain particularembodiments, the ratio of non-crosslinkable polymer to crosslinkablepolymer is about 65:35. In certain embodiments, the percentage ofcrosslinkable polymer in the hydrogel ranges from approximately 10% toapproximately 50%. In certain embodiments, the percentage ofcrosslinkable polymer in the hydrogel ranges from approximately 20% toapproximately 40%. In certain embodiments, the percentage ofcrosslinkable polymer in the hydrogel ranges from approximately 25% toapproximately 35%. In certain embodiments, the percentage ofcrosslinkable polymer in the hydrogel is aproximately 25%. In certainembodiments, the percentage of crosslinkable polymer in the hydrogel isapproximately 30%. In certain embodiments, the percentage ofcrosslinkable polymer in the hydrogel is approximately 35%. In certainembodiments, the percentage of crosslinkable polymer in the hydrogel isapproximately 40%. In certain embodiments, the percentage ofcrosslinkable polymer in the hydrogel is approximately 25%,approximately 26%, approximately 27%, approximately 28%, approximately29%, approximately 30%, approximately 31%, approximately 32%,approximately 33%, approximately 34%, or approximately 35%.

The crosslinkable polymer of the semi-interpenetrating network ofpolymer is cross-linked via a free radical mediated process. The two ormore polymeric components are mixed together in the desired proportionin the hydrogel, and a cross-linking reaction is initiated to cross-linkthe crosslinkable polymer. In certain embodiments, the polymer iscross-linked using a free radical initiator. The initiator may be athermal initiator or a photoinitiator. In certain embodiments, thepolymer is cross-linked by photo-induced cross-linking (e.g., UV light,visible light, IR light). In certain embodiments, the light is centeredat approximately 365 nm. In other embodiments, the polymer iscross-linked by heat (e.g., 30-200° C.). In other embodiments, thepolymer is cross-linked using a biological or chemical catalyst. Thecross-linking process is performed under conditions suitable to yieldthe desired properties of the resulting hydrogel. For example, theextent of cross-linking may be controlled by the time of the reaction,the amount/concentration of initiator, the polymer starting material,the initiator, the frequency of the light used to effect thecross-linking, additives, temperature of the reaction, solvent used,concentration of polmyer starting material, oxygen inhibition, waterinhibition, etc.

Typically, the initiator decomposes upon heating or exposure to acertain wavelength of light to yield two free radicals that initiate thecross-linking reaction. The initiator may work in a variety of organicsolvents , water, or aqueous solutions. Organic solvents that can beused include acetone, ethers, bezene, THF, toluene, hexanes, DMSO, DMF,etc. In certain embodiments, the cross-linking reaction is performed inwater or an aqueous solution. In certain particular embodiments, thecross-linking reaction is performed in phosphate-buffered salinesolution. The aqueous solution may be acidic or basic.

The initiator is typically chosen based on a variety of concernsincluding the structure of the polymer, the desired cross-linkedmaterial to be produced, the extent of cross-linking, the subsequent useof the material, etc. These and other concerns may be taken into accountby one of skill in the art choosing the thermal initiator to be used.The initiator may be obtained from a commercial source such asSigma-Aldrich, Ciba-Geigy, Sartomer, etc. The initiator may also beprepared synthetically.

In certain embodiments, the initiator is a thermal initiator. Anythermal initiator may be used in the cross-linking reaction. In certainembodiments, the thermal initiator is designed to work at a temperatureranging from 30° C. to 200° C. In certain embodiments, the initiator isdesigned to work at a temperature ranging from 50° C. to 170° C. Inother embodiments, the initiator is designed to work at a temperatureranging from 50° C. to 100° C. In certain embodiments, the initiator isdesigned to work at a temperature ranging from 100° C. to 170° C. Incertain partiular embodiments, the initiator is designed to work atapproximately 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, or170° C. The thermal initiators may be peroxides, peracids, peracetates,persulfates, etc. Examplary thermal initiators include tert-amylperoxybenzoate; 4,4-azobis (4-cyanovaleric acid);1,1′-azobis(cyclohexanecarbonitrile); 2,2′-azobisisobutyronitrile(AIBN); benzoyl peroxide; 2,2-bis(tert-butylperoxy)butane;1,1-bis(tert-butylperoxy)cyclohexane;2,5-bis(tert-butylperoxy)-2,5-dimethylhexane;2,5-bis(tert-butylperoxy)-2,5-dimethyl-3-hexyne;bis(1-(tert-butylperoxy)-1-methylethyl)benzene;1,1-bis(tert-butylperoxy)-3,3,5-trimethylcyclohexane; tert-butylhydroperoxide; tert-butyl peracetate; tert-butyl peroxide; tert-butylperoxybenzoate; tert-butylperoxy isopropyl carbonate; cumenehydroperoxide; cyclohexanone peroxide; dicumyl peroxide; lauroylperoxide; 2,4-pentanedione peroxide; peracetic acid; and potassiumpersulfate. In certain embodiments, a combination of thermal initiatorsis used.

In other embodiments, the initiator is a photoinitiator. Photoinitiatorsproduce reactive free radical species that initiate the cross-linking ofthe cross-linkable component of the hydrogel. Any photoinitiator may beused in the cross-linking reaction. Photoinitiated polymerizations andphotoinitiators are discussed in detail in Rabek, Mechanisms ofPhotophysical Processes and Photochemical Reactions in Polymers, NewYork: Wiley & Sons, 1987; Fouassier, Photoinitiation,Photopolymerization, and Photocuring, Cincinnati, Ohio: Hanser/Gardner;Fisher et al., “Photoinitiated Polymerization of Biomaterials” Annu.Rev. Mater. Res. 31:171-81, 2001; incorporated herein by reference. Thephotoinitiator may be designed to produce free radicals at anywavelength of light. In certain embodiments, the photoinitiator isdesigned to work using UV light (200-400 nm). In certain embodiments,long UV rays are used. In other embodiments, short UV rays are used. Inother embodiments, the photoinitiator is designed to work using visiblelight (400-800 nm). In certain embodiments, the photoinitiator isdesigned to work using blue light (420-500 nm). In yet otherembodiments, the photinitiator is designed to work using IR light(800-2500 nm). In certain embodiments, the photoinitiator is a peroxide(e.g., ROOR′). In other embodiments, the photoinitiator is a ketone(e.g., RCOR′). In other embodiments, the compound is an azo compound(e.g., compounds with a —N═N— group). In certain embodiments, thephotoinitiator is an acylphosphineoxide. In other embodiments, thephotoinitiator is a sulfur-containing compound. In still otherembodiments, the initiator is a quinone. Exemplary photoinitiatorsinclude acetophenone; anisoin; anthraquinone; anthraquinone-2-sulfonicacid, sodium salt monohydrate; (benzene)tricarbonylchromium; benzin;benzoin; benzoin ethyl ether; benzoin isobutyl ether; benzoin methylether; benzophenone; benzophenone/1-hydroxycyclohexyl phenyl ketone;3,3′,4,4′-benzophenonetetracarboxylic dianhydride; 4-benzoylbiphenyl;2-benzyl-2-(dimethylamino)-4′-morpholinobutyrophenone;4,4′-bis(diethylamino)benzophenone; 4,4′-bis(dimethylamino)benzophenone;camphorquinone; 2-chlorothioxanthen-9-one;(cumene)cyclopentadienyliron(II) hexafluorophosphate; dibenzosuberenone;2,2-diethoxyacetophenone; 4,4′-dihydroxybenzophenone;2,2-dimethoxy-2-phenylacetophenone; 4-(dimethylamino)benzophenone;4,4′-dimethylbenzil; 2,5-dimethylbenzophenone; 3,4-dimethylbenzophenone;diphenyl(2,4,6-trimethylbenzoyl)phosphineoxide/2-hydroxy-2-methylpropiophenone; 4′-ethoxyacetophenone;2-ethylanthraquinone; ferrocene; 3′-hydroxyacetophenone;4′-hydroxyacetophenone; 3-hydroxybenzophenone; 4-hydroxybenzophenone;1-hydroxycyclohexyl phenyl ketone; 2-hydroxy-2-methylpropiophenone;2-methylbenzophenone; 3-methylbenzophenone; methybenzoylformate;2-methyl-4′-(methylthio)-2-morpholinopropiophenone; phenanthrenequinone;4′-phenoxyacetophenone; thioxanthen-9-one; triarylsulfoniumhexafluoroantimonate salts; triarylsulfonium hexafluorophosphate salts;hydrogen peroxide; benzoyl peroxide; benzoin;2,2-dimethoxy-2-phenylacetophenone; dibenzoyl disulphides;diphenyldithiocarbonate; 2,2′-azobisisobutyronitrile (AIBN);camphorquinone (CQ); eosin; dimethylaminobenzoate (DMAB);dimethoxy-2-phenyl-acetophenone (DMPA); Quanta-cure ITX photosensitizer(Biddle Sawyer); Irgacure 907 (Ciba Geigy); Irgacure 651 (Ciba Geigy);Irgacure 2959 (Ciba Geigy); Darocur 2959 (Ciba Geigy);ethyl-4-N,N-dimethylaminobenzoate (4EDMAB);1-[-(4-benzoylphenylsulfanyl)phenyl]-2-methyl-2-(4-methylphenylsulfonyl)propan-1-one;1-hydroxy-cyclohexyl-phenyl-ketone;2,4,6-trimethylbenzoyldiphenylphosphine oxide;2-ethylhexyl-4-dimethylaminobenzoate;2-hydroxy-2-methyl-1-phenyl-1-propanone; 65%(oligo[2-hydroxy-2-methyl-1-[4-(1-methylvinyl)phenyl]propanone] and 35%propoxylated glyceryl triacrylate; benzil dimethyl ketal; benzophenone;blend of benzophenone and a-hydroxy-cyclohexyl-phenyl-ketone; blend ofEsacure KIP150 and Esacure TZT; blend of Esacure KIP150 and Esacure TZT;blend of Esacure KIP150 and TPGDA; blend of phosphine oxide, EsacureKIP150 and Esacure TZT; difunctional a-hydroxy ketone; ethyl4-(dimethylamino)benzoate; isopropyl thioxanthone; liquid blend of4-methylbenzophenone and benzophenone; oligo(2-hydroxy-2 methyl-1-4(1-methylvinyl)phenyl propanone (emulsion); oligo(2-hydroxy-2-methyl-1-4(1-methylvinyl)phenyl propanone and2-hydroxy-2-methyl-1-phenyl-1-propanone (monomeric); oligo(2-hydroxy-2-methyl-1-4 (1-methylvinyl)phenyl propanone and2-hydroxy-2-methyl-1-phenyl-1-propanone(polymeric);trimethylbenzophenone and methylbenzophenone; and water emulsion of2,4,6-trimethylbenzoylphosphine oxide, alpha hydroxyketone,trimethylbenzophenone, and 4-methyl benzophenone. In certainembodiments, the photoinitiator is Irgacure 2959. In certainembodiments, a combination of photoinitiators is used.

The hydrogel may be optionally purified and/or otherwise processed afterit has been prepared. In certain embodiments, after the hydrogel iscreated, it is sheared to create a hydrogel composition of the desiredelastic shear modulus. Shearing is typically done by forcing thehydrogel through a narrowed opening. In certain embodiments, smaller andsmaller openings may be used. In certain embodiments, the hydrogel isforced through a series of needles with smaller and smaller bores. Forexample, the hydrogel may be passed successively through 16 gauge, 18gauge, 20 gauge, and 22 gauge needles. In certain embodiments, asyringe-like device that can contain a larger volume is used. In otherembodiments, ultrasonic and/or mechanical shearing methods may be used.In certain embodiments, a homogenizer is used. In certain embodiments, amicrofluidizer is used. The hydrogel is typically processed until thethe desired elastic shear modulus of the material is achieved. Incertain embodiments, the elastic shear modulus ranges from approximately15 Pa to appoximately 35 Pa. In certain embodiments, the elastic shearmodulus ranges from approximately 20 Pa to appoximately 30 Pa. Incertain embodiments, the elastic shear modulus is approximately 21 Pa.In certain embodiments, the elastic shear modulus is approximately 22Pa. In certain embodiments, the elastic shear modulus is approximately23 Pa. In certain embodiments, the elastic shear modulus isapproximately 24 Pa. In certain embodiments, the elastic shear modulusis approximately 25 Pa. In certain embodiments, the elastic shearmodulus is approximately 26 Pa.

In certain embodiments, the hydrogel is a composition comprisingacrylated polyethylene glycol and polyethylene glycol. In certainparticular embodiments, the hydrogel is a compostion comprisingpolyethylene glycol diacrylate and polyethylene glycol. In certainembodiments, the molecular weight of the polyethylene glycol diacrylateis approximately 10,000 g/mol. In certain embodiments, the molecularweight of the polyethylene glycol is approximately 10,000 g/mol. Sevenparts of a 10% solution of the non-crosslinkable polymer is mixed withthree parts of a 10% solution of the crosslinkable polymer, and theresulting composition is cross-linked using a photoinitiator and UVlight. In certain embodiments, the photoinitiator Irgacure 2959 (CibaSpecialty Chemicals, Tarrytown, N.J.) is used in the photopolymerizationreaction. The intensity of the UV light ranges from about 1 mW/cm² toabout 20 mW/cm². In certain embodiments, the intensity of the UV lightis about 10 mW/cm². The resulting hydrogel is sheared by passing itthrough needles of descreasing bore size (e.g., 16 gauge, 18 gauge, 20gauge, and 22 gauge needles). In certain embodiments, the hydrogel ispassed through each size of needle twice before using a smaller needle.

In certain embodiments, the hydrogel is a composition comprisingacrylated polyethylene glycol and hyaluronic acid. In certain particularembodiments, the hydrogel is a compostion comprising polyethylene glycoldiacrylate and hyaluronic acid. In certain embodiments, the molecularweight of the polyethylene glycol diacrylate is approximately 10,000g/mol. In certain embodiments, the molecular weight of the hyaluronicacid is approximately 560,000 g/mol. Seventy-three parts of a 1 mg/mLsolution of the hyaluronic acid is mixed with twenty-seven parts of a100 mg/mL of the crosslinkable polymer, and the resulting composition iscross-linked using a photoinitiator and UV light. In certainembodiments, the photoinitiator Irgacure 2959 (Ciba Specialty Chemicals,Tarrytown, N.J.) is used in the photopolymerization reaction. Theintensity of the UV light ranges from about 0.5 mW/cm² to about 20mW/cm². In certain embodiments, the intensity of the UV light rangesfrom about 1 mW/cm²to about 5 mW/cm². In certain embodiments, theintensity of the UV light ranges from about 5 mW/cm² to about 10 mW/cm².In certain embodiments, the intensity of the UV light is about 1 mW/cm².In certain embodiments, the intensity of the UV light is about 2 mW/cm².In certain embodiments, the intensity of the UV light is about 5 mW/cm².In certain embodiments, the intensity of the UV light is about 10mW/cm². The resulting hydrogel is sheared by passing it through needlesof descreasing bore size (e.g., 16 gauge, 18 gauge, 20 gauge, and 22gauge needles). In certain embodiments, the hydrogel is passed througheach size of needle twice before using a smaller needle.

In certain embodiments, the hydrogel is a composition comprisingacrylated polyethylene glycol and dextran. In certain particularembodiments, the hydrogel is a compostion comprising polyethylene glycoldiacrylate and dextran. In certain embodiments, the molecular weight ofthe polyethylene glycol diacrylate is approximately 10,000 g/mol. Incertain embodiments, the molecular weight of the dextran isapproximately 200,000 g/mol. Seven parts of a 20 mg/mL solution of thedextran is mixed with three parts of a 100 mg/mL of the crosslinkablepolymer, and the resulting composition is cross-linked using aphotoinitiator and UV light. In certain embodiments, the photoinitiatorIrgacure 2959 (Ciba Specialty Chemicals, Tarrytown, N.J.) is used in thephotopolymerization reaction. The intensity of the UV light ranges fromabout 1 mW/cm² to about 20 mW/cm². In certain embodiments, the intensityof the UV light is about 10 mW/cm². The resulting hydrogel is sheared bypassing it through needles of descreasing bore size (e.g., 16 gauge, 18gauge, 20 gauge, and 22 gauge needles). In certain embodiments, thehydrogel is passed through each size of needle twice before using asmaller needle.

The hydrogel useful in the present invention typically do not degrade invivo or breakdown slowly. The lack of biodegradability is predominantlyuseful to prevent the hydrogel having to be re-injected into the vocalfolds or other area repeatedly. Typically, such injections should berepeated only once per month, once every 2-3 months, once every 6months, or once every year. The longer the time between injections ofthe hydrogel the better.

The hydrogel may be combined with other therapeutically active agentsand/or pharmaceutically acceptable excipients to form a compositionuseful for vocal cord repair or other soft tissue repair oraugmentation.

In some embodiments, the present invention provides for compositionscomprising hydrogels as described herein. Such compositions mayoptionally comprise one or more additional biologically active agents.In some embodiments, inventive compositions are administered to humans.

Although the descriptions of hydrogel compositions provided herein areprincipally directed to compositions which are suitable foradministration to humans, it will be understood by the skilled artisanthat such compositions are generally suitable for administration toanimals of all sorts. Modification of pharmaceutical compositionssuitable for administration to humans in order to render thecompositions suitable for administration to various animals is wellunderstood, and the ordinarily skilled veterinary pharmacologist candesign and/or perform such modification with merely ordinary, if any,experimentation. Subjects to which administration of the pharmaceuticalcompositions of the invention is contemplated include, but are notlimited to, humans and/or other primates; mammals, including mammalssuch as cattle, pigs, horses, sheep, cats, ferrets, and/or canines.

The formulations of the pharmaceutical compositions described herein maybe prepared by any method known or hereafter developed in the art ofpharmaceutics. In general, such preparatory methods include the step ofbringing the hydrogel into association with one or more excipientsand/or one or more other accessory ingredients, and then, if necessaryand/or desirable, shaping, and/or packaging the product into a desiredsingle-or multi-dose unit.

The relative amounts of the hydrogel, the pharmaceutically acceptableexcipient(s), and/or any additional ingredients in a pharmaceuticalcomposition of the invention will vary, depending upon the identity,size, and/or condition of the subject. By way of example, thecomposition may comprise between 1% and 99% (w/w) of the hydrogel.

Pharmaceutical formulations of the present invention may additionallycomprise a pharmaceutically acceptable excipient, which, as used herein,includes any and all solvents, dispersion media, diluents, or otherliquid vehicles, dispersion or suspension aids, surface active agents,isotonic agents, thickening or emulsifying agents, preservatives, solidbinders, lubricants and the like, as suited to the particular dosageform desired. Remington's The Science and Practice of Pharmacy, 21^(st)Edition, A. R. Gennaro, (Lippincott, Williams & Wilkins, Baltimore, Md.,2006; incorporated herein by reference) discloses various excipientsused in formulating pharmaceutical compositions and known techniques forthe preparation thereof. Except insofar as any conventional excipient isincompatible with a substance or its derivatives, such as by producingany undesirable biological effect or otherwise interacting in adeleterious manner with any other component(s) of the pharmaceuticalcomposition, its use is contemplated to be within the scope of thisinvention.

In some embodiments, the pharmaceutically acceptable excipient is atleast 95%, 96%, 97%, 98%, 99%, or 100% pure. In some embodiments, theexcipient is approved for use in humans and for veterinary use. In someembodiments, the excipient is approved by United States Food and DrugAdministration. In some embodiments, the excipient is pharmaceuticalgrade. In some embodiments, the excipient meets the standards of theUnited States Pharmacopoeia (USP), the European Pharmacopoeia (EP), theBritish Pharmacopoeia, and/or the International Pharmacopoeia.

Pharmaceutically acceptable excipients used in the manufacture of thehydrogel compositions include, but are not limited to, inert diluents,dispersing agents, surface active agents and/or emulsifiers,disintegrating agents, preservatives, buffering agents, lubricatingagents, and/or oils. Such excipients may optionally be included in theinventive formulations. Excipients such as coloring agents can bepresent in the composition, according to the judgment of the formulator.

Exemplary diluents include, but are not limited to, calcium carbonate,sodium carbonate, calcium phosphate, dicalcium phosphate, calciumsulfate, calcium hydrogen phosphate, sodium phosphate lactose, sucrose,cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol,inositol, sodium chloride, dry starch, cornstarch, powdered sugar, etc.,and combinations thereof

Exemplary dispersing agents include, but are not limited to, potatostarch, corn starch, tapioca starch, sodium starch glycolate, clays,alginic acid, guar gum, citrus pulp, agar, bentonite, cellulose and woodproducts, natural sponge, cation-exchange resins, calcium carbonate,silicates, sodium carbonate, cross-linked poly(vinyl-pyrrolidone)(crospovidone), sodium carboxymethyl starch (sodium starch glycolate),carboxymethyl cellulose, cross-linked sodium carboxymethyl cellulose(croscarmellose), methylcellulose, pregelatinized starch (starch 1500),microcrystalline starch, water insoluble starch, calcium carboxymethylcellulose, magnesium aluminum silicate (Veegum), sodium lauryl sulfate,quaternary ammonium compounds, etc., and combinations thereof.

Exemplary surface active agents and/or emulsifiers include, but are notlimited to, natural emulsifiers (e.g. acacia, agar, alginic acid, sodiumalginate, tragacanth, chondrux, cholesterol, xanthan, pectin, gelatin,egg yolk, casein, wool fat, cholesterol, wax, and lecithin), colloidalclays (e.g. bentonite [aluminum silicate] and Veegum [magnesium aluminumsilicate]), long chain amino acid derivatives, high molecular weightalcohols (e.g. stearyl alcohol, cetyl alcohol, oleyl alcohol, triacetinmonostearate, ethylene glycol distearate, glyceryl monostearate, andpropylene glycol monostearate, polyvinyl alcohol), carbomers (e.g.carboxy polymethylene, polyacrylic acid, acrylic acid polymer, andcarboxyvinyl polymer), carrageenan, cellulosic derivatives (e.g.carboxymethylcellulose sodium, powdered cellulose, hydroxymethylcellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose,methylcellulose), sorbitan fatty acid esters (e.g. polyoxyethylenesorbitan monolaurate [Tween®20], polyoxyethylene sorbitan [Tween®60],polyoxyethylene sorbitan monooleate [Tween®80], sorbitan monopalmitate[Span®40], sorbitan monostearate [Span®60], sorbitan tristearate[Span®65], glyceryl monooleate, sorbitan monooleate [Span®80]),polyoxyethylene esters (e.g. polyoxyethylene monostearate [Myrj®45],polyoxyethylene hydrogenated castor oil, polyethoxylated castor oil,polyoxymethylene stearate, and Solutol), sucrose fatty acid esters,polyethylene glycol fatty acid esters (e.g. Cremophor®), polyoxyethyleneethers, (e.g. polyoxyethylene lauryl ether [Brij®30]),poly(vinyl-pyrrolidone), diethylene glycol monolaurate, triethanolamineoleate, sodium oleate, potassium oleate, ethyl oleate, oleic acid, ethyllaurate, sodium lauryl sulfate, Pluronic F 68, Poloxamer 188,cetrimonium bromide, cetylpyridinium chloride, benzalkonium chloride,docusate sodium, etc. and/or combinations thereof.

Exemplary binding agents include, but are not limited to, starch (e.g.cornstarch and starch paste); gelatin; sugars (e.g. sucrose, glucose,dextrose, dextrin, molasses, lactose, lactitol, mannitol,); natural andsynthetic gums (e.g. acacia, sodium alginate, extract of Irish moss,panwar gum, ghatti gum, mucilage of isapol husks,carboxymethylcellulose, methylcellulose, ethylcellulose,hydroxyethylcellulose, hydroxypropyl cellulose, hydroxypropylmethylcellulose, microcrystalline cellulose, cellulose acetate,poly(vinyl-pyrrolidone), magnesium aluminum silicate (Veegum), and larcharabogalactan); alginates; polyethylene oxide; polyethylene glycol;inorganic calcium salts; silicic acid; polymethacrylates; waxes; water;alcohol; etc.; and combinations thereof.

Exemplary preservatives may include antioxidants, chelating agents,antimicrobial preservatives, antifungal preservatives, alcoholpreservatives, acidic preservatives, and other preservatives. Exemplaryantioxidants include, but are not limited to, alpha tocopherol, ascorbicacid, acorbyl palmitate, butylated hydroxyanisole, butylatedhydroxytoluene, monothioglycerol, potassium metabisulfite, propionicacid, propyl gallate, sodium ascorbate, sodium bisulfite, sodiummetabisulfite, and sodium sulfite. Exemplary chelating agents includeethylenediaminetetraacetic acid (EDTA), citric acid monohydrate,disodium edetate, dipotassium edetate, edetic acid, fumaric acid, malicacid, phosphoric acid, sodium edetate, tartaric acid, and trisodiumedetate. Exemplary antimicrobial preservatives include, but are notlimited to, benzalkonium chloride, benzethonium chloride, benzylalcohol, bronopol, cetrimide, cetylpyridinium chloride, chlorhexidine,chlorobutanol, chlorocresol, chloroxylenol, cresol, ethyl alcohol,glycerin, hexetidine, imidurea, phenol, phenoxyethanol, phenylethylalcohol, phenylmercuric nitrate, propylene glycol, and thimerosal.Exemplary antifungal preservatives include, but are not limited to,butyl paraben, methyl paraben, ethyl paraben, propyl paraben, benzoicacid, hydroxybenzoic acid, potassium benzoate, potassium sorbate, sodiumbenzoate, sodium propionate, and sorbic acid. Exemplary alcoholpreservatives include, but are not limited to, ethanol, polyethyleneglycol, phenol, phenolic compounds, bisphenol, chlorobutanol,hydroxybenzoate, and phenylethyl alcohol. Exemplary acidic preservativesinclude, but are not limited to, vitamin A, vitamin C, vitamin E,beta-carotene, citric acid, acetic acid, dehydroacetic acid, ascorbicacid, sorbic acid, and phytic acid. Other preservatives include, but arenot limited to, tocopherol, tocopherol acetate, deteroxime mesylate,cetrimide, butylated hydroxyanisol (BHA), butylated hydroxytoluened(BHT), ethylenediamine, sodium lauryl sulfate (SLS), sodium lauryl ethersulfate (SLES), sodium bisulfite, sodium metabisulfite, potassiumsulfite, potassium metabisulfite, Glydant Plus®, Phenonip®,methylparaben, Germall 115, Germaben II, Neolone™, Kathon™, and Euxyl®.In certain embodiments, the preservative is an anti-oxidant. In otherembodiments, the preservative is a chelating agent.

Exemplary buffering agents include, but are not limited to, citratebuffer solutions, acetate buffer solutions, phosphate buffer solutions,ammonium chloride, calcium carbonate, calcium chloride, calcium citrate,calcium glubionate, calcium gluceptate, calcium gluconate, D-gluconicacid, calcium glycerophosphate, calcium lactate, propanoic acid, calciumlevulinate, pentanoic acid, dibasic calcium phosphate, phosphoric acid,tribasic calcium phosphate, calcium hydroxide phosphate, potassiumacetate, potassium chloride, potassium gluconate, potassium mixtures,dibasic potassium phosphate, monobasic potassium phosphate, potassiumphosphate mixtures, sodium acetate, sodium bicarbonate, sodium chloride,sodium citrate, sodium lactate, dibasic sodium phosphate, monobasicsodium phosphate, sodium phosphate mixtures, tromethamine, magnesiumhydroxide, aluminum hydroxide, alginic acid, pyrogen-free water,isotonic saline, Ringer's solution, ethyl alcohol, etc., andcombinations thereof.

Exemplary lubricating agents include, but are not limited to, magnesiumstearate, calcium stearate, stearic acid, silica, talc, malt, glycerylbehanate, hydrogenated vegetable oils, polyethylene glycol, sodiumbenzoate, sodium acetate, sodium chloride, leucine, magnesium laurylsulfate, sodium lauryl sulfate, etc., and combinations thereof.

Exemplary oils include, but are not limited to, almond, apricot kernel,avocado, babassu, bergamot, black current seed, borage, cade, camomile,canola, caraway, carnauba, castor, cinnamon, cocoa butter, coconut, codliver, coffee, corn, cotton seed, emu, eucalyptus, evening primrose,fish, flaxseed, geraniol, gourd, grape seed, hazel nut, hyssop,isopropyl myristate, jojoba, kukui nut, lavandin, lavender, lemon,litsea cubeba, macademia nut, mallow, mango seed, meadowfoam seed, mink,nutmeg, olive, orange, orange roughy, palm, palm kernel, peach kernel,peanut, poppy seed, pumpkin seed, rapeseed, rice bran, rosemary,safflower, sandalwood, sasquana, savoury, sea buckthorn, sesame, sheabutter, silicone, soybean, sunflower, tea tree, thistle, tsubaki,vetiver, walnut, and wheat germ oils. Exemplary oils include, but arenot limited to, butyl stearate, caprylic triglyceride, caprictriglyceride, cyclomethicone, diethyl sebacate, dimethicone 360,isopropyl myristate, mineral oil, octyldodecanol, oleyl alcohol,silicone oil, and combinations thereof.

Liquid dosage forms for parenteral administration include, but are notlimited to, pharmaceutically acceptable emulsions, microemulsions,solutions, suspensions, syrups, and elixirs. In addition to thehydrogel, the liquid dosage forms may comprise inert diluents commonlyused in the art such as, for example, water or other solvents,solubilizing agents and emulsifiers such as ethyl alcohol, isopropylalcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzylbenzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils(in particular, cottonseed, groundnut, corn, germ, olive, castor, andsesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycolsand fatty acid esters of sorbitan, and mixtures thereof. In certainembodiments, the hydrogel of the invention is mixed with solubilizingagents such as Cremophor®, alcohols, oils, modified oils, glycols,polysorbates, cyclodextrins, polymers, and combinations thereof.

Injectable formulations, for example, sterile injectable aqueous oroleaginous suspensions, may be formulated according to the known artusing suitable dispersing or wetting agents and suspending agents. Asterile injectable preparation may be a sterile injectable solution,suspension, or emulsion in a nontoxic parenterally acceptable diluent orsolvent, for example, as a solution in 1,3-butanediol. Among theacceptable vehicles and solvents that may be employed are water,Ringer's solution, U.S.P. and isotonic sodium chloride solution. Inaddition, sterile, fixed oils are conventionally employed as a solventor suspending medium. For this purpose any bland fixed oil can beemployed including synthetic mono-or diglycerides. In addition, fattyacids such as oleic acid are used in the preparation of injectables.

Injectable formulations can be sterilized, for example, by filtrationthrough a bacterial-retaining filter, or by incorporating sterilizingagents in the form of sterile solid compositions which can be dissolvedor dispersed in sterile water or other sterile injectable medium priorto use.

General considerations in the formulation and/or manufacture ofpharmaceutical agents may be found, for example, in Remington: TheScience and Practice of Pharmacy 21^(st) ed., Lippincott Williams &Wilkins, 2005.

The inventive hydrogel may be combined with one or more biologicallyactive agents. In certain embodiments, the hydrogel is combined with apharmaceutical agent. In certain embodiments, the hydrogel is combinedwith an anti-inflammatory agent. In certain embodiments, the hydrogel iscombined with an anti-fibrotic agent. In certain embodiments, thehydrogel is combined with an anti-proliferative agent. In certainembodiments, the hydrogel is combined with an antibiotic. In certainembodiments, the hydrogel is combined with a protein or peptide. Incertain embodiments, the hydrogel is combined with a growth factor. Incertain embodiments, the hydrogel is combined with cells. In certainembodiments, the hydrogel is combined with autologous cells. In certainembodiments, the hydrogel is combined with stem cells. In certainembodiments, the hydrogel is combined with a liquid excipient foradministering the hydrogel. In certain embodiments, the excipient is anaqueous solution. In certain embodiments, the excipient is a bufferedaqueous solution. In certain embodiments, the excipient isphosphate-buffered saline solution. In certain embodiments, theexcipient is isotonic with extracellular fluid.

Uses

The invention further provides methods of injecting polymeric hydrogelsor compositions thereof into the vocal cords or just under the phonatoryepithelium of the vocal cords to restore the pliability of scarred vocalfolds. In certain embodiments, the hydrogel is injected in the lostsubepithelial superficial lamina propria layer of the mucosa. Theinventive hydrogels may also be used to repair or augment other softtissues. In certain embodiments, the hydrogel is used to augment thebladder neck for urinary incontinence. In other embodiments, thehydrogel is used as dermal fillers, breast implants, intervertebraldisks, muscle-mass, and joint fluid.

In the setting of vocal cord repair, the hydrogel or other material isinjected into the residual superificial lamina propria or just underphonatory epithelium. As described herein, it has been discovered thathydrogels with an elastic shear modulus ranging from approximately 20 Pato approximately 30 Pa are particularly useful in restoring thepliability of the phonatory mucosa. One or both vocal folds may betreated using the inventive technique. In certain embodiments,approximately 0.1 mL to approximately 5 mL of hydrogel composition isinjected into the vocal fold. The hydrogel is injected into the vocalfolds using a very thin gauge needle (e.g., 25-30 gauge).

The hydrogel may last from weeks to months in the subepithelial regionor superficial lamina propria (SLP) of the vocal cords. As needed, theprocedure may be repeated in order to restore the pliability of thevocal cords, thereby restoring the patient's voice. The frequency oftreatment will depend on the patient and the hydrogel being injected. Inthe case, of a patient with vocal fold paralysis, the treating physicianmay desire that the implant degrade after approximately 6 months toprovide voice function while allowing for natural vocal nerveregeneration.

Other materials with similar properties (e.g., elastic shear modulus ofapproximately 15 Pa to approximately 35 Pa) to those of the hydrogelsdescribed herein may also be used in vocal cord or soft tissue repair oraugmentation. Typically the material is a soft gel-like material. Suchmaterials may be used alone or in conjunction with the hydrogelsdescribed herein. Exemplary materials for use in the inventive methodsmay include viscosupplements and dermal fillers. In certain embodiments,the material used comprises hyaluronic acid or a salt thereof. Incertain embodiments, the viscosupplement is HYALGAN® (sodiumhyaluronate), SYNVISC® (Hylan G-F 20), or ORTHOVISC® (high molecularweight hyaluronan). Other viscosupplements may be used. In certainembodiments, the dermal filler is RESTYLANE® (hyaluronic acid), PERLANE®(hyaluronic acid), HYLAFORM® (stabilized hyaluronic acid), or RADIESSE®(calcium hydroxylapatite microspheres in a water-based gel). Otherdermal fillers may be used.

Kits

The invention also provides packages or kits, comprising one or morehydrogels or hydrogel components as described herein in a container. Forexample, the container may include a hydrogel composition ready for usein a patient. Or the containers may contain the components of thehydrogel (e.g., crosslinkable polymer, non-crosslinkable polymer) whichmust be mixed and cross-linked to form the hydrogel. The package canalso include a notice associated with the container, typically in a formprescribed by a government agency regulating the manufacture, use, orsale of medical devices and/or pharmaceuticals, whereby the notice isreflective of approval by the agency of the compositions, for human orveterinary administration to treat vocal cord disease or other softtissue repair or augmentation. Instructions for the use of the hydrogelcomposition may also be included. Such instructions may includeinformation relating to administration of the hydrogel to a patient. Inparticular, the instructions may include information regarding theinjection of the hydrogel into the vocal cords of patient.

In certain embodiments of the invention the kit will include multipleindividual containers, each containing a component of the hydrogel. Forexample, a first container may contain a crosslinkable polymer, and asecond container may contain a non-crosslinkable polymer. Thecross-linking initiator may be provided in yet a third container. Thepolymers may be provided in predetermined amounts such that when mixedwith each other in solution in the presence of an initiator they form ahydrogel having the desired characteristics. The package may alsoinclude one or more containers containing biologically active agent(s)to be included in the hydrogel prior to administration.

The package may include a device or receptacle for preparation of ahydrogel composition. The device may be, e.g., a measuring or mixingdevice.

The package may also optionally include a device for administering ahydrogel composition of the invention. Exemplary devices includespecialized syringes, needles, and catheters that are compatible with avariety of laryngoscope designs.

The components of the kit may be provided in a single larger container,e.g., a plastic or styrofoam box, in relatively close confinement.Typically, the kit is conveniently packaged for use by a health careprofessional. In certain embodiments, the components of the kit aresterilely packaged for use in a sterile environment such as an operatingroom or physician's office.

Examples Example 1 Preparation of Hydrogels for Injection into a ScarredVocal Fold

Semi-interpenetrating networks technology was used to make the hydrogelsof this Example. This process involves using a cross-linkable polymer(X) that is polymerized in the presence of a non-crosslinkable polymer(Y). The physicochemical properties of the gels were controlled byindependently varying i) the fraction (f) of X in the solution; ii) theconcentrations (C_(X) and C_(Y)) of the components; and iii) themolecular weights (M_(X) and M_(Y)) of the components of the hydrogel.All the polymers used to form the hydrogels in this Example werewater-soluble. Photopolymerization using UV light (centered at 365 nm)was carried out using Irgacure 2959 (Ciba Specialty Chemicals,Tarrytown, N.Y.) as the photoinitiator. The description given belowoutlines the detailed protocol for preparing certain exemplary hydrogelswhere,

X=Polyethylene Glycol Diacrylate (PEG-DA) obtained from SunBio Inc.

Y=Polyethylene Glycol (PEG)

C_(X)=100 mg/mL (10% w/v) before mixing

C_(Y)=100 mg/mL (10% w/v) before mixing

M_(X)=10,000 (10 kDa)

M_(Y)=10,000 (10 kDa)

f=0.3

Protocol

-   1. Make separate solutions of PEG-DA and PEG (100 mg/mL both) in    phosphate-buffered saline (PBS). Make a solution of Irgacure 2959    (50 mg/mL) in an ethanol solution (70% v/v) in deionized water.-   2. Add 700 μL of the PEG solution to a well in a 12-well plate. Add    300 μL of PEG-DA solution to the PEG solution and mix well. Add 10    μL of Irgacure 2959 solution to the solution of PEG and PEG-DA.-   3. Place the 12-well plate under a UV light source from EXFO UV    curing system. The sides of the apparatus are covered in aluminum    foil to not only protect the observers from the UV light but also to    reflect the UV light to the inside. The intensity of the light    falling at the bottom of the plate is adjusted to 10 mW/cm² by    manipulating the intensity knob and the height of the light source    from the bottom of the plate.-   4. The plate is then placed so that the UV beam emanating from the    light source is centered at the center of the well containing the    above-prepared solution. The UV light is shone for 200 s at which    point the gelation is complete.-   5. The gel thus prepared is then incubated in PBS (˜8 mL) at 37° C.    in one of the wells of a 6-well plate for 24 hours. A biological    incubator at a temperature of 37° C. and an atmosphere of 5% CO₂ is    used for the incubation.-   6. After the incubation is complete, the gel is put in the bore of a    Luer-lok syringe (3 mL or 5 mL). The gel is sheared through the    opening of the syringe into another Luer-lok syringe (3 mL or 5 mL),    and the process is repeated to get the hydrogel back into the first    syringe.-   7. The above shearing process is repeated using needles of    decreasing bore size progressively. Specifically, 16 gauge, 18    gauge, 20 gauge, and 22 gauge needles are used successively with the    hydrogels sheared through each needle twice.-   8. The hydrogel thus obtained at the end of the shearing process is    added into the bore of an unused, new Luer-lok syringe (3 mL or 5    mL) and capped with an unused, new 16 gauge needle. The    gel-containing syringe can be stored at room temperature for a    period of 24-48 hours. Storage at 4° C. is recommended for long-term    use.-   9. If sterile hydrogels are to be prepared, the entire gelling    apparatus is moved into a biological hood. Disinfection is carried    out by cleaning the equipments with a 70% ethanol solution (v/v).    Sterile technique is used for handling the hydrogels and other    equipments. The following modifications are made to the    above-described protocol when making sterile hydrogels:    -   a. The solutions of the as-obtained polymers are made using        sterile PBS in autoclaved vials and are not filtered through        syringe filters. The photoinitiator solution, on the other hand,        is filtered through a syringe filter (0.2 μm) before use.    -   b. Hydrogels are formed in sterile 12-well plates and handled        using autoclaved forceps.    -   c. Once the hydrogel is formed using the above mentioned        procedure, the hydrogel is further disinfected by incubation in        a 70% ethanol solution (v/v) made in sterile deionized water.        The hydrogel is incubated for 1 min in 8 mL of 70% ethanol        placed in a well of a sterile 6-well plate. The incubation is        repeated a total of 3 times using fresh ethanol solution every        time.    -   d. The hydrogel disinfected by incubation in 70% ethanol is then        incubated in sterile PBS (8 mL) in a well of a sterile 6-well        plate for 5 min. The incubation is repeated a total of 3 times        using fresh sterile PBS every time to remove the ethanol        absorbed by the gel. Higher incubation times may also be used if        the gel seems to have retained more alcohol (the gel look        shriveled upon absorption of alcohol).    -   e. After removing the ethanol, the hydrogel is incubated in        fresh, sterile PBS (8 mL) in a new sterile 6-well plate for 24        hours. The PBS in the well is replaced after 12 hours of        incubation with fresh sterile PBS (8 mL) by removing the gel        (using autoclaved forceps) and putting it in a new sterile        6-well plate containing sterile PBS.    -   f. The hydrogel is then sheared using the procedure outlined in        steps 6 and 7 above. The only difference is that the shearing is        carried out using sterile syringes and needles and inside a        biological hood.-   10. We have prepared several different gels using the procedure    outlined above along with independently and systematically changing    X, Y, f, C_(X), C_(Y), M_(X), and M_(Y). e.g.:    -   a. X=Polyethylene Glycol Diacrylate from SunBio Inc.        -   Y=Hyaluronic Acid from Genzyme        -   M_(X)=10 kDa        -   M_(Y)=560 kDa        -   C_(X)=100 mg/mL        -   C_(Y)=1 mg/mL        -   f=0.27    -   b. X=Polyethylene Glycol Diacrylate from SunBio Inc.        -   Y=Dextran        -   M_(X)=10 kDa        -   M_(Y)=200 kDa        -   C_(X)=100 mg/mL        -   C_(Y)=20 mg/mL        -   f=0.3

Other hydrogels that have been prepared using the above protocol arelisted in the table below.

Volumetric Elastic fraction of Shear Cross-linkable Non-crosslinkable Xbefore Modulus Name component (X) component (Y) gelation (f) G′ (Pa)PEG-HA PEG-diacrylate Hyaluronic Acid 0.3 22 Pa (10 kDa) from (HA; 74kDa) from SunBio LifeCore Conc.: 100 mg/mL Biomedical Conc.: 1 mg/mLPEG-Dextran PEG-diacrylate Dextran (100 to 200 kDa) 0.3 24 Pa (10 kDa)from from Sigma SunBio Conc.: 20 mg/mL Conc.: 100 mg/mL PEG-AlginatePEG-diacrylate Sodium alginate 0.28 25 Pa (10 kDa) from (viscosity:20,000 SunBio to 40,000 cps) from Conc.: 100 mg/mL Aldrich Conc.: 0.5mg/mL PEG-Polylysine PEG-diacrylate Poly-L-Lysine (70 0.28 21 Pa (10kDa) from to 150 kDa) from SunBio Fluka Biochemika Conc.: 100 mg/mL HAmethacrylate- HA methacrylate Hyaluronic Acid 0.7 24 Pa HA synthesizedusing (HA; 64 kDa) from HA (75 kDa) from LifeCore LifeCore BiomedicalBiomedical Conc.: 20 mg/mL Conc.: 20 mg/mL

Example 2 Demonastration of the Efficacy of Hydrogels in Repairing thePliability of the Phonatory Mucosa using Ex Vivo Models

Cow Larynx Model. An ex vivo bovine larynx model was used to theevaluate effects of gel stiffness on mucosal wave amplitude, as ameasure of vocal fold pliability. Adult cow cadaver larynges wereprepared by cutting a 1 cm by 3 cm window in the thyroid lamina and thenremoving a block of the thyroarytenoid muscle to expose the deep surfaceof the vocal ligament, a layer of collagenous tissue between the SLP andthe thyroarytenoid muscle. The ligament was opened with microscissorsand the soft contents of the lamina propria were carefully removed overthe entire extent of the vocal fold, leaving only the thin andtransparent epithelium with minimal attached SLP. The test materialswere layered behind the epithelium in volumes equal to the volume of theremoved lamina propria (˜0.25 ml), resulting in a layer of gel 2-3 mmthick. An oval piece of stiff latex sheet was placed behind the testmaterial in the location previously occupied by the vocal ligament. Theremaining cavity through the muscle and thyroid cartilage was thenfilled with stiff alginate for measurement of mucosal wave amplitudeusing high speed imaging. The alginate, dam, and test material wereeasily removed for sequential testing of different hydrogels in the samebiomechanical environment. PEG30 (G′=25 Pa), PEG34 (G′=121 Pa), andPEG50 (G′=566 Pa) were prepared and tested. As a control, the vocal foldwas also filled only with the alginate to create a stiff condition toensure that an insignificant amount of SLP remained. All materials weretested in the same larynx with each material tested twice. Multiple highspeed video clips from tests controlled for subglottal pressure wereselected and maximum mid-membranous vocal fold excursion was measured ina blinded manner by two observers. VF excursion was highly correlatedwith driving pressure for each of the gels (FIG. 3), and this model wassensitive to gel stiffness. As shown in FIG. 3, PEG30 supported morevocal fold excursion than the stiffer gels, with the alginate-filledcondition showing very little movement.

Testing of vocal-fold implant materials biomechanically equivalent toPEG30. Different hydrogels biomechanically similar to PEG30 (as judgedby measuring their elastic shear modulus, G′) were prepared bysystematically varying the concentration, volumetric ratio in theprecursor solution, and the polymer used for the crosslinkable andnon-crosslinkable component. We were able to identify five materialsthat may be considered mechanically equivalent to PEG30 based on elasticshear modulus (see table in Example 1). These materials were also testedin the cow larynx model using the procedure outlined above. In additionto the hydrogels and alginate we also tested RESTYLANE® (hyaluronicacid), a commercially available dermal filler that has been used forapplication in the vocal folds. All the materials were tested in asingle cow larynx with each material being tested twice. Multiple highspeed video clips from tests controlled for sub-glottal pressure wereselected and maximum mid-membranous vocal fold excursion was measuredusing a MATLAB program.

FIG. 4 shows the maximum vocal fold (VF) excursion for the differentmaterials at the lowest driving pressure that was able to producevibration in the VFs. It is important to note that the VF excursion incase of Restylane and PEG-HA were measured at driving pressures of 13.9cm and 9.7 cm, respectively while those for the other materials weremeasured at pressures of approximately 6.5 cm. A high value of thelowest driving pressure required to initiate phonation indicates acomparatively higher stiffness for the material being tested than thosewith lower values of the lowest driving pressure.

All the materials developed in our labs supported VF excursions greaterthan that of alginate or Restylane. In addition, the VF excursionsupported by the materials was similar in magnitude thereby suggestingthat all of these materials are biomechanically suitable for repairingthe phonatory mucosa of the vocal folds.

Example 3 Demonstration of Safety and Efficacy of PEG30 in an In VivoModel

Testing in canines. PEG30 was injected unilaterally in sixteen normalcanine VFs with post-injection survival periods of 1, 2, 3, and 4 months(n=4/time point). An average of 60 μL of PEG30 was injected in the VF.We made periodic examinations of VF appearance and in vivo functionusing methods developed at the MGH Center for Laryngeal Surgery andVoice Rehabilitation. Stroboscopic and high speed videos of the VFvibration (4000 frames/sec) were recorded during these exams while usinga tracheal needle to inject air for phonation. These recordings allow usto assess the pliability of the VFs under physiological conditions. Highresolution MRI and histology were performed on the excised laryngesafter euthanasia to identify the location of the PEG30 in the VF and toobserve tissue responses to the injected material.

Ability of PEG30 to maintain the pliability of vocal folds in vivo. As ameasure of VF pliability, the amplitude of VF vibration was measuredfrom the high-speed videos (HSV) collected in the in vivo setting duringthe periodic exams. The ratio of amplitude of vibration on the injectedand non-injected side was compared at each in vivo testing time point.FIG. 5 shows the average percentage of amplitude ratio of thePEG30-injected VF to the non-injected VF over a period of 4 months. Eachtime point is an average of at least 3 measures obtained from a total of7 canines. There was no statistical difference between the amplituderatio measures at all time points tested thereby demonstrating minimalperturbation in pliability of the PEG30-injected VFs as compared to thenon-injected VF. In vivo examination of the canine VFs using anoperating microscope revealed little or no signs of inflammation in theanimals tested. Apart from a few animals where mild redness of theinjected VF was observed transiently in the first week or two after theinjection, the injected VF surface looked identical to the non-injectedVF under high magnification.

Histological analysis of the vocal folds showed that PEG30 generates aforeign-body reaction in the animal VFs characterized by presence ofmacrophages that actively engulf the PEG30. However, the presence ofPEG30 and the associated reaction did not compromise the vibration ofthe VFs. The PEG30 resorbed and the foreign body reaction resolvedalmost completely by the end of 16 weeks with no apparent damage to theVF pliability. Overall the injection of PEG30 in the VFs did not causeany adverse tissue reaction nor hinder the normal vibration of the VFs.These results therefore suggest the potential utility of PEG30 as avocal fold implant to repair the pliability of the phonatory mucosa.

Example 4 Incorporation of Therapeutics in the Hydrogel for Slow Releasein the Vocal Folds

Poly-L-lactic-co-glycolic acid (PLGA) nanoparticles (NPs) thatincorporate rapamycin, an anti-fibrotic drug were made using singleemulsion technique. This technique has been used to prepare numerousother drug-loaded PLGA nanoparticles. Rapamycin-loaded NPs were madeusing two PLGA polymers with different lactic:glycolic acid ratio: PLGA75:25 and PLGA 50:50 with lactic:glycolic acid ratio of 75:25 and 50:50,respectively. As shown in FIG. 6, the rate of release of rapamycin fromPLGA 75:25 NPs in an aqueous buffer is lower than that from PLGA 50:50NPs for the same amount of total drug loaded. The rate of degradation ofPLGA 75:25 in buffer is slower than that of PLGA 50:50. Consequently,rapamycin-loaded NPs made using PLGA 75:25 were expected to release thedrug slower than those made using PLGA 50:50. The rapamycin-loaded PLGA50:50 NPs approximately 200 nm in size were loaded in PEG30 hydrogels byincorporating the NPs in the precursor solution prior to gelation. PLGA50:50-PEG30 gels containing up to 8 mg/mL (in the precursor solution) ofthe rapamycin-loaded PLGA NPs had biomechanical properties similar toPEG30 not containing any NPs. This result suggests that PEG30 may beused to not only repair the pliability of the phonatory mucosa but alsorelease therapeutics such as rapamycin in the vocal folds. The processof making drug-loaded PLGA NPs is amenable to incorporation and slowrelease of a wide variety of drugs. Furthermore, the ease with whichthese NPs can be incorporated in our hydrogels without compromisingtheir favorable mechanical properties leads us to believe that a varietyof drug-eluting, biomechanically relevant hydrogels may be preparedspecifically for use in the vocal folds.

Equivalents and Scope

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. The scope of the presentinvention is not intended to be limited to the above description, butrather is as set forth in the appended claims.

In the claims articles such as “a,” “an,” and “the” may mean one or morethan one unless indicated to the contrary or otherwise evident from thecontext. Claims or descriptions that include “or” between one or moremembers of a group are considered satisfied if one, more than one, orall of the group members are present in, employed in, or otherwiserelevant to a given product or process unless indicated to the contraryor otherwise evident from the context. The invention includesembodiments in which exactly one member of the group is present in,employed in, or otherwise relevant to a given product or process. Theinvention also includes embodiments in which more than one, or all ofthe group members are present in, employed in, or otherwise relevant toa given product or process. Furthermore, it is to be understood that theinvention encompasses all variations, combinations, and permutations inwhich one or more limitations, elements, clauses, descriptive terms,etc., from one or more of the claims or from relevant portions of thedescription is introduced into another claim. For example, any claimthat is dependent on another claim can be modified to include one ormore limitations found in any other claim that is dependent on the samebase claim. Furthermore, where the claims recite a composition, it is tobe understood that methods of using the composition for any of thepurposes disclosed herein are included, and methods of making thecomposition according to any of the methods of making disclosed hereinor other methods known in the art are included, unless otherwiseindicated or unless it would be evident to one of ordinary skill in theart that a contradiction or inconsistency would arise. For example, itis to be understood that any of the compositions of the invention can beused for vocal cord repair or other soft tissue repair or augmentation.It is also to be understood that any of the compositions made accordingto the methods for preparing compositions disclosed herein can be usedfor vocal cord repair or other soft tissue repair or augmentation. Inaddition, the invention encompasses compositions made according to anyof the methods for preparing compositions disclosed herein.

Where elements are presented as lists, e.g., in Markush group format, itis to be understood that each subgroup of the elements is alsodisclosed, and any element(s) can be removed from the group. It is alsonoted that the term “comprising” is intended to be open and permits theinclusion of additional elements or steps. It should be understood that,in general, where the invention, or aspects of the invention, is/arereferred to as comprising particular elements, features, steps, etc.,certain embodiments of the invention or aspects of the inventionconsist, or consist essentially of, such elements, features, steps, etc.For purposes of simplicity those embodiments have not been specificallyset forth in haec verba herein. Thus for each embodiment of theinvention that comprises one or more elements, features, steps, etc.,the invention also provides embodiments that consist or consistessentially of those elements, features, steps, etc.

Where ranges are given, endpoints are included. Furthermore, it is to beunderstood that unless otherwise indicated or otherwise evident from thecontext and/or the understanding of one of ordinary skill in the art,values that are expressed as ranges can assume any specific value withinthe stated ranges in different embodiments of the invention, to thetenth of the unit of the lower limit of the range, unless the contextclearly dictates otherwise. It is also to be understood that unlessotherwise indicated or otherwise evident from the context and/or theunderstanding of one of ordinary skill in the art, values expressed asranges can assume any subrange within the given range, wherein theendpoints of the subrange are expressed to the same degree of accuracyas the tenth of the unit of the lower limit of the range.

In addition, it is to be understood that any particular embodiment ofthe present invention may be explicitly excluded from any one or more ofthe claims. Any embodiment, element, feature, application, or aspect ofthe compositions and/or methods of the invention, can be excluded fromany one or more claims. For purposes of brevity, all of the embodimentsin which one or more elements, features, purposes, or aspects isexcluded are not set forth explicitly herein.

1. A polymeric hydrogel comprising a semi-interpenetrating network of across-linked polymer and a water soluble polymer, wherein the hydrogelhas an elastic shear modulus ranging from about 15 Pa to about 35 Pa.2.-3. (canceled)
 4. The polymeric hydrogel of claim 1 comprising: across-linked acrylated derivative of a polymer; and a water-solublepolymer; wherein the hydrogel is a semi-interpenetrating network ofpolymers, and wherein the hydrogel has an elastic shear modulus rangingfrom about 15 Pa to about 35 Pa.
 5. The polymeric hydrogel of claim 1comprising: a cross-linked acrylated derivative of polyalkylene glycol;and a water-soluble polymer selected from the group consisting ofpolyethers, polyols, poly(amino acids), proteins, and polysaccharides;wherein the hydrogel is a semi-interpenetrating network of polymers; andwherein the hydrogel has an elastic shear modulus ranging from about 15Pa to about 35 Pa. 6.-8. (canceled)
 9. The polymeric hydrogel of claim 1comprising: a cross-linked acrylated derivative of polyalkylene glycol;and a cross-linked version of a water-soluble polymer selected from thegroup consisting of polyethers, polyols, poly(amino acids), proteins,peptides, polysaccharides, and elastomeric polymers; wherein thehydrogel is a semi-interpenetrating network of polymers; and wherein thehydrogel has an elastic shear modulus ranging from about 15 Pa to about35 Pa.
 10. The polymeric hydrogel of claim 1 comprising: cross-linkedacrylated derivative of polyethylene glycol; and a water-soluble polymerselected from the group consisting of polyethers, polyols, poly(aminoacids), proteins, and polysaccharides; wherein the hydrogel is asemi-interpenetrating network of polymers; and wherein the hydrogel hasan elastic shear modulus ranging from about 15 Pa to about 35 Pa. 11.The polymeric hydrogel of claim 1 comprising: cross-linked polyethyleneglycol diacrylate; and a water soluble polymer selected from the groupconsisting of polyethylene glycol (PEG), poly(lysine), hyaluronic acid(HA), dextrans, alginates, gelatins, elastins, collagens, celluloses,methylcelluloses, and derivatives thereof; wherein the hydrogel is asemi-interpenetrating network of polymers; and wherein the hydrogel hasan elastic shear modulus ranging from about 15 Pa to about 35 Pa. 12.The polymeric hydrogel of claim 1 comprising: cross-linked hyaluronicmethacrylate; and a water soluble polymer selected from the groupconsisting of polyethylene glycol (PEG), poly(lysine), hyaluronic acid(HA), dextrans, alginates, gelatins, elastins, collagens, celluloses,methylcelluloses, and derivatives thereof; wherein the hydrogel is asemi-interpenetrating network of polymers; and wherein the hydrogel hasan elastic shear modulus ranging from about 15 Pa to about 35 Pa. 13.The polymeric hydrogel of claim 1, wherein the hydrogel has an elasticshear modulus ranging from about 20 Pa to about 30 Pa.
 14. The polymerichydrogel of claim 1, wherein the hydrogel has an elastic shear modulusranging from about 20 Pa to about 25 Pa.
 15. The polymeric hydrogel ofclaim 1, wherein the hydrogel has an elastic shear modulus of about 25Pa.
 16. The polymeric hydrogel of claim 1, wherein the water-solublepolymer is selected from the group consisting of polyethylene glycol(PEG), hyaluronic acid (HA), dextran, poly(glyceraol sebacate) (PGS),methyl cellulose, collagen, elastin, gelatin, alignate, andpoly(lysine). 17.-26. (canceled)
 27. The polymeric hydrogel of claim 1,wherein the cross-linked polymer is photo-crosslinked.
 28. The polymerichydrogel of claim 1 further comprising a bioactive agent.
 29. Thepolymeric hydrogel of claim 28, wherein the bioactive agent is a cell.30. (canceled)
 31. The polymeric hydrogel of claim 28, wherein thebioactive agent is a stem cell.
 32. The polymeric hydrogel of claim 28,wherein the bioactive agent is a protein or peptide.
 33. The polymerichydrogel of claim 28, wherein the bioactive agent is a polynucleotide.34. The polymeric hydrogel of claim 28, wherein the bioactive agent is apharmaceutical agent.
 35. The polymeric hydrogel of claim 28, whereinthe bioactive agent is an anti-inflammatory agent, an anti-fibroticagent, or an anti-proliferative agent. 36.-37. (canceled)
 38. Thepolymeric hydrogel of claim 28, wherein bioactive agent is encapsulatedin a nanoparticle or microparticle.
 39. A method of preparing apolymeric hydrogel comprising steps of: providing a first cross-linkablederivative of a polymer; providing a second water soluble polymer;contacting the first cross-linkable derivative of a polymer with thesecond water-soluble polymer; and cross-linking the first cross-linkablederivative of polymer in the presence of the second water-solublepolymer to form a polymeric hydrogel; wherein the hydrogel has anelastic shear modulus ranging from about 15 Pa to about 35 Pa. 40.-67.(canceled)
 68. A method of augmenting, repairing, or replacing softtissue, the method comprising: administering a polymeric hydrogel ofclaim 1 to the soft tissue of a subject. 69.-72. (canceled)
 73. A methodof augmenting the phonatory mucosa of a vocal cord, the methodcomprising: administering the polymeric hydrogel of claim 1 into a spacecreated in the superficial lamina propria or phonatory epithelium of thevocal cord of a subject. 74.-90. (canceled)